Enzymes

ABSTRACT

The present invention relates to enzymes and processes. In particular, there is described an isolated polypeptide comprising the amino acid sequence corresponding to  Citrobacter freundii  phytase or a homologue, a modified form, a functional equivalent or an effective fragment thereof. There is also described a host cell transformed or transfected with a nucleic acid encoding a bacterial phytase enzyme or a modified form as well as the use of such a phytase or modified form in food or animal feed.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/696,162, filed Apr. 3, 2007 which is a continuation-in-part ofInternational Patent Application PCT/IB2005/003660 filed Oct. 4, 2005which published as WO 2006/038128 on Apr. 13, 2006, and which claimspriority to International Patent Application PCT/IB2005/000598 filedFeb. 15, 2005, and Great Britain Patent Application No. 0422052.1 filedOct. 4, 2004.

Each of the above referenced applications, and each document cited inthis text (“application cited documents”) and each document cited orreferenced in each of the application cited documents, and anymanufacturer's specifications or instructions for any products mentionsin this text and in any document incorporated into this text, are herebyincorporated herein by reference; and, technology in each of thedocuments incorporated herein by reference can be used in the practiceof this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. Patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the inventions, and they exclude ingredients orsteps for the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. Patent law, namely, that these terms are closed ended.

The present invention relates to phytases, nucleotide sequences forsame, methods of production of phytases and their use.

FIELD OF THE INVENTION

The present invention relates to the field of enzymes for additives tofeedstuffs. More specifically, the present invention relates to phytaseswhich can be used for enhancing phosphate digestion in foods and animalfeeds.

TECHNICAL BACKGROUND AND PRIOR ART

Phytate is the major storage form of phosphorus in cereals and legumes.However, monogastric animals such as pig, poultry and fish are not ableto metabolise or absorb phytate (or phytic acid) and therefore it isexcreted leading to phosphorous pollution in areas of intense livestockproduction. Moreover phytic acid also acts as an antinutritional agentin monogastric animals by chelating metal agents such as calcium, copperand zinc.

In order to provide sufficient phosphates for growth and health of theseanimals, inorganic phosphate is added to their diets. Such addition canbe costly and further increases pollution problems.

Phytate is converted by phytases which generally catalyse the hydrolysisof phytate to lower inositol-phosphates and inorganic phosphate.Phytases are useful as additives to animal feeds where they improve theavailability of organic phosphorus to the animal and decrease phosphatepollution of the environment (Wodzinski R J, Ullah A H. Adv ApplMicrobiol. 42, 263-302 (1996)).

A number of phytases of fungal (Wyss M. et al. Appl. Environ. Microbiol.65 (2), 367-373 (1999); Berka R. M. et al. Appl. Environ. Microbiol. 64(11), 4423-4427 (1998); Lassen S. et al. Appl. Environ. Microbiol. 67(10), 4701-4707 (2001)) and bacterial (Greiner R. et al Arch. Biochem.Biophys. 303 (1), 107-113 (1993); Kerovuo et al. Appl. Environ.Microbiol. 64 (6), 2079-2085 (1998); Kim H. W. et al. Biotechnol. Lett.25, 1231-1234 (2003); Greiner R. et al. Arch. Biochem. Biophys. 341 (2),201-206 (1997); Yoon S. J. et al. Enzyme and microbial technol. 18,449-454 (1996); Zinin N. V. et al. FEMS Microbiol. Lett. 236, 283-290(2004))) origin have been described in the literature.

However, to date, none of these phytases display the properties requiredfor effective use as an animal feed supplement. In particular, fungalphytases tend to be proteolytically unstable (Igbasan F. A. et al. Arch.Anim Nutr. 53, 353-373 (2000)) and therefore susceptible to degradation,while most bacterial phytases have a narrow substrate specificity forphytate alone and degrade poorly inositol phosphates of intermediatedegrees of phosphorylation (Greiner R. et al., Arch. Biochem. Biophys.303 (1), 107-113 (1993); Kerovuo J et al. Biochem. J. 352, 623-628(2000)).

Accordingly, there is a need for improved phytases.

SUMMARY OF THE INVENTION

In a broad aspect, the present invention relates to phytases derivedfrom a bacterium and modified forms thereof. In particular the inventionrelates to wild type phytases derived from the bacterium, Citrobacterfreundii, and variant/modified forms thereof showing improvedcharacteristics compared to the wild-type enzyme.

The present invention is advantageous as it provides for novel phytasesthat have properties making them particularly useful and efficient asfeed enzymes. In particular the invention relates to isolated and/orpurified novel phytase polypeptide as described herein or functionalfragments or variants or modified forms thereof. The invention alsoprovides the nucleic acid and amino acid sequences encoding saidphytases.

To be efficient as an enzyme additive to food or animal feed, a phytasehas to combine a number of different properties. In order to be able todegrade phytic acid in the acidic environment of an animal's stomach ithas to be active at low pH, preferably over a broad range of pH values.In addition, it has to have high specific activity and preferably highthermostability to enable the protein to withstand high temperaturescommonly used in preparation of feedstuffs such as feed pellets.

It is also important that the enzyme has broad substrate specificityallowing it to hydrolyse not only phytate but also intermediate productsof phytate degradation such as inositol pentaphosphates, tetraphosphatesand triphosphates. Studies on phytate degradation in pigs show thatthese inositol oligophosphates otherwise remain largely insoluble in thesmall and large intestine and thus inaccessible to alkaline phosphatasesproduced by the animal and gut microflora (Schlemmer U. et al. Arch.Anim Nutr. 55, 255-280 (2001)). Variations in substrate specificityprofiles of different enzymes have been identified. For example,inositol-triphosphates generated by the phytase from B. subtilis areessentially resistant to further hydrolysis by this enzyme (Kerovuo J.et al. Biochem J. (200) 352, 623-628).

In another aspect of the invention there is provided a plasmid or avector system or a transformed or a transgenic organism comprising anovel phytase as described herein or a modified form thereof.

In another aspect the present invention relates to transgenic organismsmodified to express a novel phytase as described herein or a modifiedform thereof and therefore being capable of producing a phytase. Thepresent invention further provides means and methods for thebiotechnological production of phytases and their use as feedsupplements.

Aspects of the present invention are presented in the claims and in thefollowing commentary.

For ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

As used with reference to the present invention, the terms “produce”,“producing”, “produced”, “produceable”, “production” are synonymous withthe respective terms “prepare”, “preparing”, “prepared”, “preparation”,“generated”, “generation” and “preparable”.

As used with reference to the present invention, the terms “expression”,“expresses”, “expressed” and “expressable” are synonymous with therespective terms “transcription”, “transcribes”, “transcribed” and“transcribable”.

As used with reference to the present invention, the terms“transformation” and “transfection” refer to a method of introducingnucleic acid sequences into hosts, host cells, tissues or organs.

Other aspects concerning the nucleotide sequences which can be used inthe present invention include: a construct comprising the sequences ofthe present invention; a vector comprising the sequences for use in thepresent invention; a plasmid comprising the sequences for use in thepresent invention; a transformed cell comprising the sequences for usein the present invention; a transformed tissue comprising the sequencesfor use in the present invention; a transformed organ comprising thesequences for use in the present invention; a transformed hostcomprising the sequences for use in the present invention; a transformedorganism comprising the sequences for use in the present invention. Thepresent invention also encompasses methods of expressing the nucleotidesequence for use in the present invention using the same, such asexpression in a host cell; including methods for transferring same. Thepresent invention further encompasses methods of isolating thenucleotide sequence, such as isolating from a host cell.

Other aspects concerning the amino acid sequences for use in the presentinvention include: a construct encoding the amino acid sequences for usein the present invention; a vector encoding the amino acid sequences foruse in the present invention; a plasmid encoding the amino acidsequences for use in the present invention; a transformed cellexpressing the amino acid sequences for use in the present invention; atransformed tissue expressing the amino acid sequences for use in thepresent invention; a transformed organ expressing the amino acidsequences for use in the present invention; a transformed hostexpressing the amino acid sequences for use in the present invention; atransformed organism expressing the amino acid sequences for use in thepresent invention. The present invention also encompasses methods ofpurifying the amino acid sequences for use in the present inventionusing the same, such as expression in a host cell; including methods oftransferring same, and then purifying said sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS PAGE analysis of the recombinant phytase from C.freundii P3-42 purified by DEAE-Sepharose chromatography. The figurepresents the scanning trace of a digital photographic image of the lanecontaining a sample of the C. freundii phytase.

FIG. 2 shows pH profile of the phytase from C. freundii P3-42

FIG. 3 shows substrate specificity of the purified recombinant phytasefrom C. freundii P3-42 with inositol phosphate fractions of differentdegree of phosphorylation and model substrates. Abbreviations:IP6—phytic acid, IP5, IP4 and IP3—mixtures of isomeric inositol penta-,tetra- and triphosphates respectively. Fru P2—fructose 1,6-diphosphate,Fru P1—fructose 6-phosphate.

SEQ ID NO: 1 lists the sequence obtained for identification of thebacterial strain.

SEQ ID NO: 2 lists the sequence comprising the phytase gene from C.freundii P3-42.

SEQ ID NO: 3 lists the amino acid sequence of the phytase gene C.freundii P3-42.

SEQ ID NO:8 lists the amino acid sequence of the phytase gene C.freundii P3-42 with possible amino acid substitutions marked.

DETAILED DISCLOSURE OF INVENTION

The present invention features an enzyme comprising the amino acidsequence corresponding to Citrobacter freundii phytase or a modifiedform, a variant, a functional equivalent or an effective fragmentthereof.

The term “phytase” means a protein or polypeptide which is capable ofcatalysing the hydrolysis of esters of phosphoric acid including phytateand releasing inorganic phosphate. Phytases are capable to hydrolyse, inaddition to phytate, at least some of the inositol-phosphates ofintermediate degrees of phosphorylation.

The term “corresponding to Citrobacter freundii phytase” means that theenzyme need not have been obtained from a source of Citrobacterfreundii. Instead, the enzyme has to have essentially the samefunctional characteristics or sequence as that of Citrobacter freundiiphytase.

The term “functional equivalent thereof” means that the enzyme has tohave essentially the same functional characteristics as that ofwild-type Citrobacter freundii phytase. The term “modified form” or“variant” means that the enzyme has been modified from its original formbut retains essentially the same enzymatic functional characteristics asthat of wild-type Citrobacter freundii phytase. In particular, the terms“variant” or “modified form” encompass phytase enzymes with an aminoacid sequence derived from the amino acid sequence of theparent/wild-type phytase and having one or more amino acidsubstitutions, insertions, and/or deletions, which together are referredto as mutations. Modified forms or variants may be altered in the enzymecharacteristics compared to the parent enzyme. Preferably, modifiedforms or variants have an increased thermostability, an increased pepsinstability, an increased specific activity, a broader substratespecificity, or other modifications that are beneficial for theapplication of the enzyme. The term “functional” or “effective” fragmentmeans a fragment or portion of the Citrobacter freundii phytase thatretains essentially the same enzymatic function or effect.

Preferably the enzyme of this aspect of the present invention has thesame sequence or a sequence that is at least 75% identical (homologous)to that of Citrobacter freundii phytase.

Suitably, the enzyme comprises the amino acid sequence as shown in SEQID NO: 3 or a sequence having at least 75% identity (homology) theretoor a functional fragment thereof. In a preferred embodiment, theinvention provides an isolated and/or purified polypeptide having theamino acid sequence as set out in SEQ ID NO: 3 or a sequence having atleast 75% identity (homology) thereto or an effective fragment thereof.

In another embodiment, the phytase is characterised in that it isderived from Citrobacter freundii strain P3-42 deposited under accessionnumber NCIMB 41247 on Sep. 22, 2004, under the terms of the BudapestTreaty, with the National Collections of Industrial, Marine and FoodBacteria (NCIMB) in Bucksburn, Aberdeen AB21 9YA, Scotland, UK.Deposited microorganism(s) will be irrevocably and without restrictionor condition released to the public during the effective term of anypatent issued from this application.

In a preferred embodiment, the invention relates to a phytase inaccordance with any embodiment of the first aspect of the invention thatcomprises one or more mutations at the following positions (numberingaccording to the numbering in SEQ ID No. 3): 22, 23, 24, 28, 46, 53, 57,67, 74, 75, 77, 78, 79, 82, 88, 95, 96, 97, 98, 101, 102, 103, 105, 109,112, 122, 126, 136, 140, 142, 143, 148, 151, 152, 154, 156, 160, 161,164, 168, 170, 176, 177, 195, 199, 203, 204, 205, 206, 207, 215, 224,225, 229, 233, 235, 274, 279, 288, 301, 307, 308, 322, 343, 358, 360,362, 365, 366, 367, 370, 383, 384, 385, 386, 391, 393, 395, 397, 408 and414.

These positions are characterized in that mutagenesis of the enzyme atthese positions lead to an improvement in the desired enzymecharacteristics.

Preferred mutations include:

A22T, E23K, E23Q, E24D, M28L, K46E, K46R, D53K, D53N, D57Y, G67R, G74R,E75K, E75V, V77I, S78T, E79V, Q82H, Q82K, Q82R, F88Y, N95D, N95P, N96P,N96S, N96Y, Q97T, T98G, T98P, S101F, P102L, G103E, V105I, A109T, D112V,D112Y, F122Y, L126I, Y136N, E140V, K142R, T143I, T143P, N148D, K151G,M152K, M152V, T154I, S156T, L160F, K161N, N164D, E168D, A170T, L176Q,L176V, Y177F, S195T, T199I, T203I, T203L, T203S, T203W, E204A, E204G,E204H, E204I, E204N, E204R, E204V, K205P, K205R, S206R, S206T, T207A,T207S, L215F, D224H, N225D, N225E, P229S, S233C, S235A, Q274H, Q274L,Q279E, R288M, L301S, E307Y, N308D, N308T, A322V, G343A, K358R, K360N,T362A, T362I, N365D, T366S, D367N, Q370H, D383V, I384F, I384L, I384M,Q385R, P386Q, K391N, A393P, K395T, D397N, S4081 and L414I orconservative mutations at each position.

By “conservative mutations” is meant mutations to amino acid residuesthat are conservative in terms of the amino acid characteristicscompared to the amino acid residue indicated. Amino acid characteristicsinclude the size of the residue, hydrophobicity, polarity, charge,pK-value, and other amino acid characteristics known in the art and alsodescribed in more detail below.

In a particularly preferred embodiment, the mutations are at one or moreof the following positions:

23, 46, 53, 75, 82, 88, 95, 96, 98, 112, 143, 152, 176, 177, 199, 203,204, 205, 225, 229, 233, 274, 288, 307, 308, 362, 370, 384 and 385

Preferred mutations at these specific positions include:

E23K, E23Q, K46E, K46R, D53K, D53N, E75K, E75V, Q82H, Q82K, Q82R, F88Y,N95D, N95P, N96P, N96S, N96Y, T98G, D112V, D112Y, T143I, T143P, M152K,M152V, L176Q, L176V, Y177F, T199I, T203I, T203L, T203S, T203W, E204A,E204G, E204H, E204I, E204N, E204R, E204V, K205P, K205R, N225D, N225E,P229S, S233C, Q274H, Q274L, R288M, E307Y, N308D, N308T, T362A, T362I,Q370H, I384F, I384L, I384M and Q385R or conservative mutations at eachposition.

In one embodiment, there is provided a phytase comprising one mutationselected from the group consisting of:

P229S; D112V; Q82R; Q274H; D112Y; F88Y; K46E; S233C; R288M; I384L;Q385R; Q274L; E307Y; T199I; Q82K and T203I.

In a further preferred embodiment, there is provided a phytasecomprising a combination of mutations selected from the group consistingof:

K46E/Q82H; Q82K/V105I; N148D/T362I; K46E/L414I; F88Y/Y136N; T154I/P386Q;N95P/N96S; N95P/N96P; Q97T/T98G; D224H/N225E; Y177F/T199I; Q274L/Q370H;K46E/N96Y; N148D/L301S; E24D/R288M; E140V/A322V; K46E/S195T; E75K/N365D;T98P/S235A; L160F/L215F; Q274L/K395T; G67R/Q279E/N308T;K161N/P229S/R288M; D53N/D57Y/M152V; F122Y/S156T/P229S;T199I/S206R/T207S; E23K/K46E/Q82H; K46E/Q82H/Q385R; T203W/E204N/K205R;T203W/E204H/K205R; T203W/E204R/K205R; T203W/E204A/K205R;A22T/K151G/N308D; E23K/E75K/F88Y; M152K/N225D/L301S; S78T/Q274L/S408I;L176Q/T199I/T366S; K46E/V77I/T203S; K46R/T199I/D367N; G74R/E204G/R288M;A22T/T199I/S206T/T207A; Q82R/F88Y/L126I/I384L; K46E/Q82H/E168D/Q274L;Q82K/T154I/Q279E/N308T; Q82R/D112V/Q274H/T362A; E24D/E79V/N95D/K360N;E23K/M28L/A109T/T143P/I384L; D53N/D57Y/T199I/P229S/R288M;K46E/Q82H/N148D/T154I/T362I; D53N/D57Y/P229S/R288M/K358R;D53N/D57Y/T154I/P229S/R288M; Y136N/T199I/T203L/E204I/K205P;E23Q/S101F/Q274L/I384M/K391N; K46E/Q82H/N95D/D112V/K142R/D383V;D53N/D57Y/M152V/P229S/R288M/A393P; D53K/D57Y/M152V/P229S/R288M/A393P;D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T;D53N/D57Y/M152V/E204V/P229S/R288M/A393P;D53N/D57Y/M152V/T154I/P229S/R288M/A393P;D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P;K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P;Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P;Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P;H18Q/D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P;Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/A393P/D397N;

D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/N164D/Q274H/T362I/I384L/A393P;

D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/Y177F/Q274H/Q279E/T362I/A393P,and;

D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/Q274L/G343A/T362I/I384L/A393P.

In a yet further preferred embodiment, there is provided a phytasecomprising a combination of mutations selected from the group consistingof:

D57Y/F88Y/N95P/Q97T/N148D/M152V/T154I/Y177F/Q274H/I384L;D53N/D57Y/F88Y/N95P/N96P/Q97T/M152V/Y177F/Q274H/Q279E/T362I/I384L;D53N/Q82K/F88Y/N96P/T98G/V105I/N148D/T154I/Q274H/T362I/I384L/P386Q;Q82R/F88Y/N96P/T98G/V105I/D112V/Y136N/N148D/T154I/Y177F/P386Q/A393P;D53N/Q82K/F88Y/N95P/N96P/T98G/Y136N/N148D/T154I/I384L/P386Q/A393P;D57Y/Q82K/F88Y/N96P/Q97T/T98G/V105I/N148D/T154I/Y177F/Q274H/I384L/P386Q/A393P;D53N/Q82K/F88Y/N95P/Q97T/T98G/D112V/Y136N/N148D/T154I/Q274H/Q279E/I384L/P386Q/A393PandD53N/D57Y/Q82R/F88Y/N95P/N96P/Q97T/T98G/V105I/Y136N/N148D/M152V/Y177F/I384L/P386Q.

Accordingly, a preferred phytase in accordance with the presentinvention is a variant consisting of the amino acid sequence listed asSEQ ID NO: 3 and having one or more of the amino acid mutations listedabove or one of the combinations of mutations listed above.

In these embodiments, the nomenclature indicates a phytase comprisingthe amino acid sequence set out in SEQ ID NO: 3 with the mutationsindicated by reference to the positions of the amino acids in SEQ ID NO:3. The nomenclature is described in more detail below.

Suitably these variants show improved characteristics with respect toany one of the following: temperature stability, pH range, pepsinstability, specific activity, substrate specificity. Suitable methodsfor determining these characteristics are disclosed herein.

In particular, the improvements in phytase characteristics are directedto the enzyme stability under food and feed processing conditions, tothe enzyme stability during stomach transit, and to the enzyme activityand stability in human or animal stomach and/or intestinal tract makingthe improved variants particularly suitable for use as feed supplements.Thus, such improvements comprise among other parameters the increase instability at elevated temperatures, preferably at temperatures above 65°C., the increase in stability against proteolytic digestion, preferablyprotease of the digestive tract, the increase in catalytic activity atlow pH, preferably catalytic activity below pH 5.5, and the generalefficiency of releasing phosphate groups from phytate.

Suitably, in one embodiment, the phytase or functional equivalent of thepresent invention is characterised in that said phytase has a specificactivity of 1000 U/mg or higher wherein said specific activity isdetermined by incubating said phytase in a solution containing 2 mMphytate, 0.8 mM CaCl₂ in 200 mM sodium acetate buffer at pH 3.5. Inanother embodiment, the phytase of the present invention or functionalequivalent thereof may also suitably be characterised in that saidphytase has two activity maxima around pH 3 and pH 4-4.5 wherein saidactivity is determined by incubating said phytase in a solutioncontaining 2 mM phytate, 0.8 mM CaCl₂ in 200 mM sodium acetate buffer.

In a further embodiment the invention provides a method of preparing aphytase enzyme variant, which method comprises:

a) Selecting a parent phytase enzyme, wherein the parent phytase enzymeis selected from

i. a parent phytase enzyme with at least 75% homology to SEQ ID No 3

ii. a parent phytase enzyme derived from Citrobacter spp.

b) Making at least one alteration which is an insertion, a deletion or asubstitution of an amino acid residue in the parent phytase enzyme toobtain a phytase enzyme variant

c) Screening for a phytase enzyme variant which compared to the parentphytase enzyme has:

i. higher thermal stability and/or

ii. specific activity and/or

iii. proteolytic stability and/or

d) Preparing the phytase enzyme variant

In a further embodiment the invention provides a method of preparing aphytase enzyme variant, which method comprises:

a) Subjecting DNA sequence encoding a parent phytase enzyme tomutagenesis, wherein the parent phytase enzyme is selected from

i. a parent phytase enzyme with at least 75% homology to SEQ ID No 3

ii. a parent phytase enzyme derived from Citrobacter spp.

b) Expressing the mutated DNA sequence obtained in step (A) in a hostcell, and

c) Screening for host cells expressing a for a phytase enzyme variantwhich compared to the parent phytase enzyme has:

iv. higher thermal stability and/or

v. higher specific activity* and/or

vi. higher proteolytic stability and/or

Preparing the phytase enzyme variant expressed by the host cell

In the above embodiments of the invention, which relate to methods ofpreparing phytase enzyme variant the phytase enzyme variant ispreferably screened for higher thermal stability.

In the above embodiments of the invention, which relate to methods ofpreparing phytase enzyme variant the phytase enzyme variant ispreferably screened for higher thermal stability and higher proteolyticstability.

In the above embodiments of the invention, which relate to methods ofpreparing phytase enzyme variant the phytase enzyme variant ispreferably screened for higher thermal stability and higher proteolyticstability and higher specific activity.

The parent phytase enzyme is preferably derived from Citrobacterfreundii, more preferably Citrobacter freundii P3-42.

In methods of preparing a phytase enzyme variant, which method comprisessubjecting DNA sequence encoding a parent phytase enzyme to mutagenesis,the DNA sequence encoding a parent phytase enzyme is preferablysubjected to random mutagenesis, more preferably error prone PCR, evenmore preferably error threshold PCR.

The preferred methods of mutagensis of DNA sequence encoding a parentphytase enzyme is error prone PCR, more preferably error threshold PCR,other methods of mutagensis may be used either in place of errorprone/threshold PCR or in conjunction with error prone/threshold PCR.See Example 12 which provides references for suitable error prone PCRand error threshold PCR methods. Other methods are disclosed under the

The term ‘expression in a host cell’ when used in the context of theembodiments which refer to ‘a method of preparing a phytase enzymevariant’ is preferably defined as production of the phytase enzymevariant in a living organism, organ or cell as herein defined. However,it is considered that for the purpose of selection the phytase enzymevariants may also be produced via in vitro methods which utilise thetranscription and translation machinery isolated from of one or morecells isolated from one or more living organism. Such in vitroproduction of variant phytases on the invention can also be used forselecting preferred variant phytases. In vitro expression can suitableperformed using standard techniques. For reference please see ‘In vitroExpression Guide’ available from Promega Inc (Part# BR053).

DEFINITIONS OF VARIANT PHENOTYPES

Variants with higher thermal stability (thermal stability difference) ispreferably determined using the methods disclosed in Example 12.

The variant phytase enzyme prepared by the method of preparing phytaseenzyme variants preferably has a thermal stability difference of atleast 1.5, more preferably 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, mostpreferably at least 10.

Variants with higher proteolytic stability is preferably determined bythe methods disclosed in Example 12.

Preferably the phytase enzyme variant of the invention has a proteolyticstability of at least 45%, preferably 50%, 55%, more preferably at least60%.

Further Variant Embodiments

In a further embodiment the invention provides methods for thepreparation of an animal feed comprising a phytase enzyme variant.

a) Selecting a parent phytase enzyme, wherein the parent phytase enzymeis selected from

i. a parent phytase enzyme with at least 75% homology to SEQ ID No 3

ii. a parent phytase enzyme derived from Citrobacter spp.

b) Making at least one alteration which is an insertion, a deletion or asubstitution of an amino acid residue in the parent phytase enzyme toobtain a phytase enzyme variant

c) Screening for a phytase enzyme variant which compared to the parentphytase enzyme has:

i. higher thermal stability and/or

ii. specific activity and/or

iii. proteolytic stability and/or

d) Preparing the phytase enzyme variant

e) Adding the prepared phytase enzyme variant to an animal feed.

In a further embodiment the invention provides methods for thepreparation of an animal feed comprising a phytase enzyme variant.

a) Subjecting DNA sequence encoding a parent phytase enzyme tomutagenesis, wherein the parent phytase enzyme is selected from

iii. a parent phytase enzyme with at least 75% homology to SEQ ID No 3

iv. a parent phytase enzyme derived from Citrobacter spp.

b) Expressing the mutated DNA sequence obtained in step (A) in a hostcell, and

c) Screening for host cells expressing a for a phytase enzyme variantwhich compared to the parent phytase enzyme has:

vii. higher thermal stability and/or

viii. higher specific activity* and/or

ix. higher proteolytic stability and/or

d) Preparing the phytase enzyme variant expressed by the host cell

f) Adding the prepared phytase enzyme variant to an animal feed.

The preferred aspects of the method of preparing a phystase enzymevariant also apply to the above methods of preparing an animal feedcomprising a phystase enzyme variant.

In another aspect, the invention provides an isolated and/or purifiednucleic acid molecule or nucleotide sequence coding for the enzymecomprising the amino acid sequence corresponding to Citrobacter freundiiphytase, or a homologue thereof. Suitably said isolated and/or purifiednucleic acid molecule encodes a polypeptide comprising the amino acidsequence as shown in SEQ ID NO: 3 or a sequence having at least 75%identity (homology) thereto or an effective fragment thereof. In oneembodiment, the nucleic acid molecule encodes a polypeptide comprisingSEQ ID NO:3 and including mutations at the preferred positions listedherein or any of the specific mutations or combinations of mutationslisted herein. In another embodiment, the invention provides an isolatedand/or purified nucleic acid molecule comprising a nucleotide sequencethat is the same as, or is complementary to, or contains any suitablecodon substitutions for any of those of SEQ ID NO: 2 or comprises asequence which has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% sequence homology with SEQ ID NO: 2.

In a yet further aspect, the invention relates to a nucleotide sequenceand to the use of a nucleotide sequence shown as:

(a) the nucleotide sequence presented as SEQ ID No.2,

(b) a nucleotide sequence that is a variant, homologue, derivative orfragment of the nucleotide sequence presented as SEQ ID No. 2;

(c) a nucleotide sequence that is the complement of the nucleotidesequence set out in SEQ ID No. 2;

(d) a nucleotide sequence that is the complement of a variant,homologue, derivative or fragment of the nucleotide sequence presentedas SEQ ID No 2;

(e) a nucleotide sequence that is capable of hybridising to thenucleotide sequence set out in SEQ ID No. 2;

(f) a nucleotide sequence that is capable of hybridising to a variant,homologue, derivative or fragment of the nucleotide sequence presentedas SEQ ID No. 2;

(g) a nucleotide sequence that is the complement of a nucleotidesequence that is capable of hybridising to the nucleotide sequence setout in SEQ ID No. 2;

(h) a nucleotide sequence that is the complement of a nucleotidesequence that is capable of hybridising to a variant, homologue,derivative or fragment of the nucleotide sequence presented as SEQ IDNo. 2;

(i) a nucleotide sequence that is capable of hybridising to thecomplement of the nucleotide sequence set out in SEQ ID No.2;

(j) a nucleotide sequence that is capable of hybridising to thecomplement of a variant, homologue, derivative or fragment of thenucleotide sequence presented as SEQ ID No. 2.

The nucleotide sequence of the present invention may comprise sequencesthat encode for SEQ ID No. 3 or a variant, modified form, homologue orderivative thereof.

In particular, the invention provides a plasmid or vector systemcomprising a phytase as described herein or a homologue or derivativethereof. Preferably, the plasmid or vector system comprises a nucleicacid sequence as set out in SEQ ID No: 2 or a sequence that is at least75% homologous thereto or an effective fragment thereof. Suitably theplasmid or vector system is an expression vector for the expression ofany of the enzymes encoded by a nucleic acid sequence as set out in anyof SEQ ID No: 2 or a sequence that is at least 75% homologous(identical) thereto in a microorganism. In addition, the inventionprovides a plasmid or vector system for expression of any of themodified enzymes or variants described herein. Suitable expressionvectors are described herein.

In another aspect of the invention there is provided a host celltransformed or transfected with a nucleic acid encoding a phytase asdescribed herein.

Suitably, the host cell in accordance with this aspect of the inventioncomprises a phytase which comprises an amino acid sequence, orfunctional fragment thereof, as set out in SEQ ID NO: 3 or a sequencethat is at least 75% homologous thereto.

In a preferred embodiment, said host cell produces a phytase.

In a further aspect of the invention there is provided a host celltransformed or transfected with a nucleic acid encoding a phytase inaccordance with the invention. Preferably, the phytase is a Citrobacterfreundii phytase as described herein or a homologue or derivativethereof. Suitably, said phytase enzyme comprises an amino acid sequence,or functional fragment thereof, as set out in any of SEQ ID No: 3 or asequence that is at least 75% homologous (identical) thereto.Preferably, said host cell produces a phytase.

In one embodiment, the nucleotide sequence which can be used in thepresent invention is obtainable from (though it does not have to beactually obtained from) Citrobacter freundii, although it will berecognised that enzymes isolated and/or purified from equivalent strainsmay equally be used.

Suitably the host cell is derived from a microorganism includingbacteria and fungi, including yeast. In a particularly preferredembodiment the host cell is a prokaryotic bacterial cell. Suitablebacterial host cells include bacteria from different prokaryotictaxonomic groups including proteobacteria, including members of thealpha, beta, gamma, delta and epsilon subdivision, gram-positivebacteria such as Actinomycetes, Firmicutes, Clostridium and relatives,flavobacteria, cyanobacteria, green sulfur bacteria, green non-sulfurbacteria, and archaea. Particularly preferred are the Enterobacteriaceaesuch as Escherichia coli proteobacteria belonging to the gammasubdivision and low GC-Gram positive bacteria such as Bacillus.

Suitable fungal host cells include yeast selected from the groupconsisting of Ascomycota including Saccharomycetes such as Pichia,Hansenula, and Saccharomyces, Schizosaccharmycetes such asSchizosaccharomyces pombe and anamorphic Ascomycota includingAspergillus.

Other suitable eukaryotic host cells include insect cells such as SF9,SF21, Trychplusiani and M121 cells. For example, the polypeptidesaccording to the invention can advantageously be expressed in insectcell systems. As well as expression in insect cells in culture, phytasegenes can be expressed in whole insect organisms. Virus vectors such asbaculovirus allow infection of entire insects. Large insects, such assilk moths, provide a high yield of heterologous protein. The proteincan be extracted from the insects according to conventional extractiontechniques. Expression vectors suitable for use in the invention includeall vectors which are capable of expressing foreign proteins in insectcell lines.

Other host cells include plant cells selected from the group consistingof protoplasts, cells, calli, tissues, organs, seeds, embryos, ovules,zygotes, etc. The invention also provides whole plants that have beentransformed and comprise the recombinant DNA of the invention.

The term “plant” generally includes eukaryotic alga, embryophytesincluding Bryophyta, Pteridophyta and Spermatophyta such as Gymnospermaeand Angiospermae.

Preferably, said host cell is a microorganism. Preferred microorganismsinclude prokaryotic bacterial cells preferably, E. coli, B. subtilis andother species of the genus Bacillus, yeast, preferably, Hansenulapolymorpha and Schizosaccharomyces pombe.

In another aspect of the invention there is provided a bacterial cellstrain Citrobacter freundii P3-42 deposited by Danisco GlobalInnovation, Sokeritehtaantie 20, FIN-02460 Kantvik, Finland underaccession number NCIMB 41247. Such a cell can be incorporated directlyinto feed.

In another aspect, there is provided a method for the production ofphytases comprising transfecting a host cell with an expression vectoror plasmid in accordance with the invention, culturing said host cellunder conditions for the expression of the phytase and extracting saidphytase from the host cell culture media.

Suitably said method is for the production of a phytase comprisingexpressing an amino acid sequence as set out in SEQ ID NO: 3 or asequence having at least 75% homology thereto or an effective fragmentthereof in a host cell and extracting the secreted protein from the hostcell culture medium.

Another aspect of the invention provides a feed composition comprising aphytase in accordance with the invention. Preferably, the feedcomposition comprises a phytase at a concentration of 10-10000 U/kgfeed, preferably, 200-2000 U/kg feed, more preferably, 500-1000 U/kgfeed.

In one embodiment, the feed composition comprises a host cell inaccordance with the invention.

In a further aspect there is provided the use of a phytase in accordancewith the invention in food or animal feed.

PREFERABLE ASPECTS

Preferable aspects are presented in the accompanying claims and in thefollowing description and Examples section.

ADDITIONAL ADVANTAGES

The present invention is advantageous as it provides phytases that havea number of properties that make them particularly useful as additivesto animal feeds.

In particular, the phytases of the present invention are active at lowpH and, preferably in the range pH 2 to 5.5 with activity maxima aroundpH 3 and 4.5. Suitably the phytases of the present invention are activeat low pHs of the stomach environment.

Furthermore, the phytases of the present invention are efficientlysecreted both in the native host and during heterologous expression thusleading to more efficient production and isolation for addition to feed.

Moreover, the phytases of the present invention have a broad substratespecificity including penta-tetra, tri and di-phosphate substratesthereby increasing the total available phosphate to the animal. Thephytases of the present invention also have a high specific activity inthe region of 1000 U/mg+/−approximately 10%.

The products of the present invention may be used asadditives/supplements to foods and feed. The products may also be usefulin the commercial production of various inositol-phosphates.

Phytate/Phytic Acid/Phytases

Phytic acid (myo-inositol hexakisphosphate) is an important constituentin cereals, legumes and oilseed crops. The salt form, phytate, is themajor storage form of phosphorous in these plants.

Phytases catalyse phosphate monoester hydrolysis of phytic acid whichresults in the step-wise formation of myo-inositol pentakis-, tetrakis-,tris-, bis- and monophosphates, as well as the liberation of inorganicphosphate.

The terms “wild type phytase” or “wild type” as used herein refer to aphytase enzyme with an amino acid sequence found in nature.

The terms “phytase variant” or “variant” or “modified form” refer to aphytase enzyme with an amino acid sequence derived from the amino acidsequence of a parent phytase having one or more amino acidsubstitutions, insertions, and/or deletions, which together are referredto as “mutations”.

The terms “parent phytase” or “parent enzyme” refer to a phytase enzymefrom which a phytase variant is derived. A parent phytase can be a wildtype phytase or another phytase variant. In particular, in the presentinvention, a “parent phytase” may be derived from a Citrobacterfreundii. Suitably, the “parent phytase” is derived from Citrobacterfreundii strain P3-42 as described herein which, preferably has theamino acid sequence set out in SEQ ID NO:3.

Isolated

In one aspect, preferably the nucleotide or amino acid sequence is in anisolated form. The term “isolated” means that the sequence is at leastsubstantially free from at least one other component with which thesequence is naturally associated in nature and as found in nature.

Purified

In one aspect, preferably the nucleotide or amino acid sequence is in apurified form. The term “purified” means that the sequence is in arelatively pure state—e.g. at least about 90% pure, or at least about95% pure or at least about 98% pure.

Nucleotide Sequence

The scope of the present invention encompasses nucleotide sequencesencoding enzymes having the specific properties as defined herein.

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence, nucleic acid or polynucleotide sequence, andvariant, homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” or “nucleic acid molecule” in relation tothe present invention includes genomic DNA, cDNA, synthetic DNA, andRNA. Preferably it means DNA, more preferably cDNA sequence coding forthe present invention.

In a preferred embodiment, the nucleotide sequence when relating to andwhen encompassed by the per se scope of the present invention does notinclude the native nucleotide sequence according to the presentinvention when in its natural environment and when it is linked to itsnaturally associated sequence(s) that is/are also in its/their naturalenvironment. For ease of reference, we shall call this preferredembodiment the “non-native nucleotide sequence”. In this regard, theterm “native nucleotide sequence” means an entire nucleotide sequencethat is in its native environment and when operatively linked to anentire promoter with which it is naturally associated, which promoter isalso in its native environment. However, the amino acid sequenceencompassed by scope the present invention can be isolated and/orpurified post expression of a nucleotide sequence in its nativeorganism. Preferably, however, the amino acid sequence encompassed byscope of the present invention may be expressed by a nucleotide sequencein its native organism but wherein the nucleotide sequence is not underthe control of the promoter with which it is naturally associated withinthat organism.

Preparation of a Nucleotide Sequence

Typically, the nucleotide sequence encompassed by scope of the presentinvention or the nucleotide sequences for use in the present inventionare prepared using recombinant DNA techniques (i.e. recombinant DNA).However, in an alternative embodiment of the invention, the nucleotidesequence could be synthesised, in whole or in part, using chemicalmethods well known in the art (see Caruthers M H et al., (1980) NucAcids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res SympSer 225-232).

A nucleotide sequence encoding either an enzyme which has the specificproperties as defined herein or an enzyme which is suitable formodification may be identified and/or isolated and/or purified from anycell or organism producing said enzyme. Various methods are well knownwithin the art for the identification and/or isolation and/orpurification of nucleotide sequences. By way of example, PCRamplification techniques to prepare more of a sequence may be used oncea suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme or a partof the amino acid sequence of the enzyme is known, labelledoligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary, and then plating the transformed bacteria onto agar platescontaining a substrate for the enzyme (e.g. maltose for a glucosidase(maltase) producing enzyme), thereby allowing clones expressing theenzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Due to degeneracy in the genetic code, nucleotide sequences may bereadily produced in which the triplet codon usage, for some or all ofthe amino acids encoded by the original nucleotide sequence, has beenchanged thereby producing a nucleotide sequence with low homology to theoriginal nucleotide sequence but which encodes the same, or a variant,amino acid sequence as encoded by the original nucleotide sequence. Forexample, for most amino acids the degeneracy of the genetic code is atthe third position in the triplet codon (wobble position) (for referencesee Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN0-7167-1920-7) therefore, a nucleotide sequence in which all tripletcodons have been “wobbled” in the third position would be about 66%identical to the original nucleotide sequence however, the amendednucleotide sequence would encode for the same, or a variant, primaryamino acid sequence as the original nucleotide sequence.

Therefore, the present invention further relates to any nucleotidesequence that has alternative triplet codon usage for at least one aminoacid encoding triplet codon, but which encodes the same, or a variant,polypeptide sequence as the polypeptide sequence encoded by the originalnucleotide sequence.

Furthermore, specific organisms typically have a bias as to whichtriplet codons are used to encode amino acids. Preferred codon usagetables are widely available, and can be used to prepare codon optimisedgenes. Such codon optimisation techniques are routinely used to optimiseexpression of transgenes in a heterologous host.

Molecular Evolution

Once an enzyme-encoding nucleotide sequence has been isolated and/orpurified, or a putative enzyme-encoding nucleotide sequence has beenidentified, it may be desirable to modify the selected nucleotidesequence, for example it may be desirable to mutate the sequence inorder to prepare an enzyme having improved stability characteristics inaccordance with the present invention.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al (Biotechnology (1984)2, p 646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151).

Instead of site directed mutagenesis, such as described above, one canintroduce mutations randomly for instance using a commercial kit such asthe GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCRrandom mutagenesis kit from Clontech.

A third method to obtain novel sequences is to fragment non-identicalnucleotide sequences, either by using any number of restriction enzymesor an enzyme such as Dnase I, and reassembling full nucleotide sequencescoding for functional proteins. Alternatively one can use one ormultiple non-identical nucleotide sequences and introduce mutationsduring the reassembly of the full nucleotide sequence.

Thus, it is possible to produce numerous site directed or randommutations into a nucleotide sequence, either in vivo or in vitro, and tosubsequently screen for improved functionality of the encodedpolypeptide by various means.

As a non-limiting example, mutations or natural variants of apolynucleotide sequence can be recombined with either the wildtype orother mutations or natural variants to produce new variants. Such newvariants can also be screened for improved functionality of the encodedpolypeptide. The production of new preferred variants may be achieved byvarious methods well established in the art, for example the ErrorThreshold Mutagenesis (WO 92/18645), oligonucleotide mediated randommutagenesis (U.S. Pat. No. 5,723,323), DNA shuffling (U.S. Pat. No.5,605,793), exo-mediated gene assembly (WO 0058517), or RCR®Recombination Chain Reaction (EP 1230390 and U.S. Pat. No. 6,821,758).Other suitable methods are described, for example in WO 0134835, WO02/097130, WO 03/012100, WO03/057247, WO 2004/018674, U.S. Pat. No.6,303,344 and U.S. Pat. No. 6,132,970.

The application of the above-mentioned and similar molecular evolutionmethods allows the identification and selection of variants of theenzymes of the present invention which have preferred characteristicswithout any prior knowledge of protein structure or function, and allowsthe production of non-predictable but beneficial mutations or variants.There are numerous examples of the application of molecular evolution inthe art for the optimisation or alteration of enzyme activity, suchexamples include, but are not limited to one or more of the following:optimised expression and/or activity in a host cell or in vitro,increased enzymatic activity, altered substrate and/or productspecificity, increased or decreased enzymatic or structural stability,altered enzymatic activity/specificity in preferred environmentalconditions, e.g. temperature, pH, substrate

Amino Acid Sequences

The scope of the present invention also encompasses amino acid sequencesof enzymes having the specific properties as defined herein.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The enzyme encompassed in the present invention may be used inconjunction with other enzymes. Thus the present invention also covers acombination of enzymes wherein the combination comprises the enzyme ofthe present invention and another enzyme, which may be another enzymeaccording to the present invention. This aspect is discussed in a latersection.

Preferably the amino acid sequence when relating to and when encompassedby the per se scope of the present invention is not a native enzyme. Inthis regard, the term “native enzyme” means an entire enzyme that is inits native environment and when it has been expressed by its nativenucleotide sequence.

Variants/Homologues/Derivatives

The present invention also encompasses the use of variants, homologuesand derivatives of any amino acid sequence of an enzyme or of anynucleotide sequence encoding such an enzyme.

Here, the term “homologue” means an entity having a certain homologywith the amino acid sequences and the nucleotide sequences. Here, theterm “homology” can be equated with “identity”. Suitably, “homologous”in this context refers to the percentage of sequence identity betweentwo enzymes after aligning their sequences using alignment algorithms asdescribed in more detail below.

In the present context, a homologous amino acid sequence is taken toinclude an amino acid sequence which may be at least 75, 80, 81, 85 or90% identical, preferably at least 95, 96, 97, 98 or 99% identical tothe sequence. Typically, the homologues will comprise the same activesites etc.—e.g as the subject amino acid sequence. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

By “functional fragment” is meant a fragment of the polypeptide thatretains that characteristic properties of that polypeptide. In thecontext of the present invention, a functional fragment of a phytaseenzyme is a fragment that retains the carotenoid cleavage capability ofthe whole protein.

In the present context, an homologous nucleotide sequence is taken toinclude a nucleotide sequence which may be at least 75, 80, 81, 85 or90% identical, preferably at least 95, 96, 97, 98 or 99% identical to anucleotide sequence encoding an enzyme of the present invention (thesubject sequence). Typically, the homologues will comprise the samesequences that code for the active sites etc. as the subject sequence.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present invention it is preferred to express homologyin terms of sequence identity.

For the amino acid sequences and the nucleotide sequences, homologycomparisons can be conducted by eye, or more usually, with the aid ofreadily available sequence comparison programs. These commerciallyavailable computer programs can calculate % homology between two or moresequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc.Acids Research 12 p 387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th)Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) andthe GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 7-60).

However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups Amino acids can be groupedtogether based on the properties of their side chain alone. However itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput. Appl Biosci. 9:745-756) (Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor. Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids.

SET SUB-SET Hydrophobic F W Y H K M Aromatic F W Y H I L V A G CAliphatic I L V Polar W Y H K R E Charged H K R E D D C S T N QPositively H K R charged Negatively E D charged Small V C A G S P TinyA G S T N D

The present invention also encompasses conservative or homologoussubstitutions or mutations (substitution and replacement are both usedherein to mean the interchange of an existing amino acid residue, withan alternative residue) that may occur, i.e. like-for-like substitution.Thus, the term “conservative mutation” refers to an amino acid mutationthat a person skilled in the art would consider conservative to a firstmutation. “Conservative” in this context means conserving or invariablein terms of the amino acid characteristics. If, for example, a mutationleads at a specific position to a substitution of an aromatic amino acidresidue (e.g. Tyr) with an aliphatic amino acid residue (e.g. Leu) thena substitution at the same position with a different aliphatic aminoacid (e.g. Ile or Val) is referred to as a conservative mutation.Further amino acid characteristics include size of the residue,hydrophobicity, polarity, charge, pK-value, and other amino acidcharacteristics known in the art. Accordingly, a conservative mutationmay include substitution such as basic for basic, acidic for acidic,polar for polar etc.

Non-conservative substitution may also occur i.e. from one class ofresidue to another or alternatively involving the inclusion of unnaturalamino acids such as ornithine (hereinafter referred to as Z),diaminobutyric acid ornithine (hereinafter referred to as B), norleucineornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine,naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

Nomenclature

In the present invention, the conventional one-letter and three-lettercodes for amino acid residues are used. For ease of reference, mutationsin enzyme variants are described by use of the following nomenclature:amino acid residue in the parent enzyme; position; substituted aminoacid residue(s). According to this nomenclature, the substitution of,for instance, an alanine residue for a glycine residue at position 20 isindicated as Ala20Gly or A20G. The deletion of alanine in the sameposition is shown as Ala20* or A20*. The insertion of an additionalamino acid residue (e.g. a glycine) is indicated as Ala20AlaGly orA20AG. The deletion of a consecutive stretch of amino acid residues(e.g. between alanine at position 20 and glycine at position 21) isindicated as Δ(Ala20-Gly21) or Δ(A20-G21). When a parent enzyme sequencecontains a deletion in comparison to the enzyme sequence used fornumbering an insertion in such a position (e.g. an alanine in thedeleted position 20) is indicated as *20Ala or *20A. Multiple mutationsare separated by a plus sign or a slash. For example, two mutations inpositions 20 and 21 substituting alanine and glutamic acid for glycineand serine, respectively, are indicated as A20G+E21S or A20G/E21S. Whenan amino acid residue at a given position is substituted with two ormore alternative amino acid residues these residues are separated by acomma or a slash. For example, substitution of alanine at position 30with either glycine or glutamic acid is indicated as A20G,E or A20G/E,or A20G, A20E. When a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position. Thus, for instance, when amodification of an alanine in position 20 is mentioned but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid residue (i.e. any one of R, N, D,C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences of the present invention.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences presented herein, or anyderivative, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used as aprobe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other homologues may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other species, and probing suchlibraries with probes comprising all or part of any one of the sequencesin the attached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequences of theinvention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used toproduce a primer, e.g. a PCR primer, a primer for an alternativeamplification reaction, a probe e.g. labelled with a revealing label byconventional means using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector.

Biologically Active

Preferably, the variant sequences etc. are at least as biologicallyactive as the sequences presented herein.

As used herein “biologically active” refers to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree), and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

In particular, variant sequences or modified forms thereof have asimilar enzymatic profile to the profile of the phytase identifiedherein. This profile includes characteristics such as being a secretedprotein, having a pH optimum in the range of pH 2 to 5.5, preferably 3.0to 3.5, retaining at least 50% of the maximum activity over the pH range2.0-5.5 and/or having a specific activity over 1000 U/mg.

Hybridisation

The present invention also encompasses sequences that are complementaryto the nucleic acid sequences of the present invention or sequences thatare capable of hybridising either to the sequences of the presentinvention or to sequences that are complementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequencesthat are capable of hybridising to the sequences that are complementaryto the sequences presented herein, or any derivative, fragment orderivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising understringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

More preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridising to thenucleotide sequences presented herein under conditions of intermediateto maximal stringency.

In a preferred aspect, the present invention covers nucleotide sequencesthat can hybridise to the nucleotide sequence of the present invention,or the complement thereof, under stringent conditions (e.g. 50° C. and0.2×SSC).

In a more preferred aspect, the present invention covers nucleotidesequences that can hybridise to the nucleotide sequence of the presentinvention, or the complement thereof, under high stringent conditions(e.g. 65° C. and 0.1×SSC).

Site-Directed Mutagenesis

Once an enzyme-encoding nucleotide sequence has been isolated and/orpurified, or a putative enzyme-encoding nucleotide sequence has beenidentified, it may be desirable to mutate the sequence in order toprepare an enzyme of the present invention.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al., (Biotechnology(1984)2, p 646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151). A further method isdescribed in Sarkar and Sommer (Biotechniques (1990), 8, p 404-407—“Themegaprimer method of site directed mutagenesis”).

Recombinant

In one aspect the sequence for use in the present invention is arecombinant sequence—i.e. a sequence that has been prepared usingrecombinant DNA techniques.

These recombinant DNA techniques are within the capabilities of a personof ordinary skill in the art. Such techniques are explained in theliterature, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,Cold Spring Harbor Laboratory Press.

Synthetic

In one aspect the sequence for use in the present invention is asynthetic sequence—i.e. a sequence that has been prepared by in vitrochemical or enzymatic synthesis. It includes, but is not limited to,sequences made with optimal codon usage for host organisms—such as themethylotrophic yeasts Pichia and Hansenula.

Expression of Enzymes

The nucleotide sequence for use in the present invention may beincorporated into a recombinant replicable vector. The vector may beused to replicate and express the nucleotide sequence, in enzyme form,in and/or from a compatible host cell.

Expression may be controlled using control sequences eg. regulatorysequences.

The enzyme produced by a host recombinant cell by expression of thenucleotide sequence may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. The coding sequencesmay be designed with signal sequences which enhance direct secretion ofthe substance coding sequences through a particular prokaryotic oreukaryotic cell membrane.

Advantageously, the enzymes of the present invention are secreted.

Expression Vector

The terms “plasmid”, “vector system” or “expression vector” means aconstruct capable of in vivo or in vitro expression. In the context ofthe present invention, these constructs may be used to introduce genesencoding enzymes into host cells. Suitably, the genes whose expressionis introduced may be referred to as “expressible transgenes”.

Preferably, the expression vector is incorporated into the genome of asuitable host organism. The term “incorporated” preferably covers stableincorporation into the genome.

The nucleotide sequences described herein including the nucleotidesequence of the present invention may be present in a vector in whichthe nucleotide sequence is operably linked to regulatory sequencescapable of providing for the expression of the nucleotide sequence by asuitable host organism.

The vectors for use in the present invention may be transformed into asuitable host cell as described below to provide for expression of apolypeptide of the present invention.

The choice of vector eg. a plasmid, cosmid, or phage vector will oftendepend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or moreselectable marker genes—such as a gene, which confers antibioticresistance eg. ampicillin, kanamycin, chloramphenicol or tetracyclinresistance. Alternatively, the selection may be accomplished byco-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect, transform, transduce or infect a host cell.

Thus, in a further embodiment, the invention provides a method of makingnucleotide sequences of the present invention by introducing anucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector.

The vector may further comprise a nucleotide sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1, pIJ702 and pET11.

Regulatory Sequences

In some applications, the nucleotide sequence for use in the presentinvention is operably linked to a regulatory sequence which is capableof providing for the expression of the nucleotide sequence, such as bythe chosen host cell. By way of example, the present invention covers avector comprising the nucleotide sequence of the present inventionoperably linked to such a regulatory sequence, i.e. the vector is anexpression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme ofthe present invention may also be achieved by the selection ofheterologous regulatory regions, e.g. promoter, secretion leader andterminator regions.

Preferably, the nucleotide sequence according to the present inventionis operably linked to at least a promoter.

Examples of suitable promoters for directing the transcription of thenucleotide sequence in a bacterial, fungal or yeast host are well knownin the art.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence for use accordingto the present invention directly or indirectly attached to a promoter.

An example of an indirect attachment is the provision of a suitablespacer group such as an intron sequence, such as the Sh1-intron or theADH intron, intermediate the promoter and the nucleotide sequence of thepresent invention. The same is true for the term “fused” in relation tothe present invention which includes direct or indirect attachment. Insome cases, the terms do not cover the natural combination of thenucleotide sequence coding for the protein ordinarily associated withthe wild type gene promoter and when they are both in their naturalenvironment.

The construct may even contain or express a marker, which allows for theselection of the genetic construct.

For some applications, preferably the construct of the present inventioncomprises at least the nucleotide sequence of the present inventionoperably linked to a promoter.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that comprises either the nucleotide sequence or an expressionvector as described above and which is used in the recombinantproduction of an enzyme having the specific properties as defined hereinor in the methods of the present invention.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a nucleotide sequence that expresses theenzymes described in the present invention. The cells will be chosen tobe compatible with the said vector and may for example be prokaryotic(for example bacterial), fungal, yeast or plant cells. Preferably, thehost cells are not human cells.

Examples of suitable bacterial host organisms are gram positive or gramnegative bacterial species.

Depending on the nature of the nucleotide sequence encoding the enzymeof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or other fungimay be preferred. In general, yeast cells are preferred over fungalcells because they are easier to manipulate. However, some proteins areeither poorly secreted from the yeast cell, or in some cases are notprocessed properly (e.g. hyperglycosylation in yeast). In theseinstances, a different fungal host organism should be selected.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

The host cell may be a protease deficient or protease minus strain.

The genotype of the host cell may be modified to improve expression.

Examples of host cell modifications include protease deficiency,supplementation of rare tRNA's, and modification of the reductivepotential in the cytoplasm to enhance disulphide bond formation.

For example, the host cell E. coli may overexpress rare tRNA's toimprove expression of heterologous proteins as exemplified/described inKane (Curr Opin Biotechnol (1995), 6, 494-500 “Effects of rare codonclusters on high-level expression of heterologous proteins in E. coli”).The host cell may be deficient in a number of reducing enzymes thusfavouring formation of stable disulphide bonds as exemplified/describedin Bessette (Proc Natl Acad Sci USA (1999), 96, 13703-13708 “Efficientfolding of proteins with multiple disulphide bonds in the Escherichiacoli cytoplasm”).

In one embodiment, host cells in the context of the present inventioninclude those cells that can be added directly into animal feed.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence coding for theenzymes as described in the present invention and/or products obtainedtherefrom, and/or wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe organism.

Suitable organisms may include a prokaryote, fungus, yeast or a plant.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence coding forthe enzymes as described in the present invention and/or the productsobtained therefrom, and/or wherein a promoter can allow expression ofthe nucleotide sequence according to the present invention within theorganism. Preferably the nucleotide sequence is incorporated in thegenome of the organism.

The term “transgenic organism” does not cover native nucleotide codingsequences in their natural environment when they are under the controlof their native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence coding for the enzymes as described in the present invention,constructs according to the present invention, vectors according to thepresent invention, plasmids according to the present invention, cellsaccording to the present invention, tissues according to the presentinvention, or the products thereof.

For example the transgenic organism may also comprise the nucleotidesequence coding for the enzyme of the present invention under thecontrol of a heterologous promoter.

Transformation of Host Cells/Organism

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis.

Teachings on the transformation of prokaryotic hosts is well documentedin the art, for example see Sambrook et al (Molecular Cloning: ALaboratory Manual, 2nd edition, 1989, Cold Spring Harbor LaboratoryPress). Other suitable methods are set out in the Examples herein. If aprokaryotic host is used then the nucleotide sequence may need to besuitably modified before transformation—such as by removal of introns.

Filamentous fungi cells may be transformed using various methods knownin the art—such as a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known. The use of Aspergillus as a host microorganismis described in EP 0 238 023.

Another host organism can be a plant. A review of the general techniquesused for transforming plants may be found in articles by Potrykus (AnnuRev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27). Further teachings onplant transformation may be found in EP-A-0449375.

General teachings on the transformation of fungi, yeasts and plants arepresented in following sections.

Transformed Fungus

A host organism may be a fungus—such as a filamentous fungus. Examplesof suitable such hosts include any member belonging to the generaThermomyces, Acremonium, Aspergillus, Penicillium, Mucor, Neurospora,Trichoderma and the like.

Teachings on transforming filamentous fungi are reviewed in U.S. Pat.No. 5,741,665 which states that standard techniques for transformationof filamentous fungi and culturing the fungi are well known in the art.An extensive review of techniques as applied to N. crassa is found, forexample in Davis and de Serres, Methods Enzymol (1971) 17A: 79-143.

Further teachings on transforming filamentous fungi are reviewed in U.S.Pat. No. 5,674,707.

In one aspect, the host organism can be of the genus Aspergillus, suchas Aspergillus niger.

A transgenic Aspergillus according to the present invention can also beprepared by following, for example, the teachings of Turner G. 1994(Vectors for genetic manipulation. In: Martinelli S. D., Kinghorn J. R.(Editors) Aspergillus: 50 years on. Progress in industrial microbiologyvol 29. Elsevier Amsterdam 1994. pp. 641-666).

Gene expression in filamentous fungi has been reviewed in Punt et al.(2002) Trends Biotechnol 2002 May; 20(5):200-6, Archer & Peberdy CritRev Biotechnol (1997) 1470 17(4):273-306.

Transformed Yeast

In another embodiment, the transgenic organism can be a yeast.

A review of the principles of heterologous gene expression in yeast areprovided in, for example, Methods Mol Biol (1995), 49:341-54, and CurrOpin Biotechnol (1997) October; 8(5):554-60

In this regard, yeast—such as the species Saccharomyces cerevisi orPichia pastoris (see FEMS Microbiol Rev (2000 24(1):45-66), may be usedas a vehicle for heterologous gene expression.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

For the transformation of yeast, several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al., (1978, Proceedings of the National Academy of Sciences of theUSA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, Het al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells may be selected using various selectivemarkers—such as auxotrophic markers dominant antibiotic resistancemarkers.

Transformed Plants/Plant Cells

A host organism suitable for the present invention may be a plant. Areview of the general techniques may be found in articles by Potrykus(Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou(Agro-Food-Industry Hi-Tech Mar./Apr. 1994 17-27).

Culturing and Production

Host cells transformed with the nucleotide sequence of the presentinvention may be cultured under conditions conducive to the productionof the encoded enzyme and which facilitate recovery of the enzyme fromthe cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions and obtaining expressionof the enzyme.

The protein produced by a recombinant cell may be displayed on thesurface of the cell.

The enzyme may be secreted from the host cells and may conveniently berecovered from the culture medium using well-known procedures.

Secretion

It may be desirable for the enzyme to be secreted from the expressionhost into the culture medium from where the enzyme may be more easilyrecovered. According to the present invention, the secretion leadersequence may be selected on the basis of the desired expression host.Hybrid signal sequences may also be used with the context of the presentinvention.

Typical examples of heterologous secretion leader sequences are thoseoriginating from the fungal amyloglucosidase (AG) gene (glaA—both 18 and24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeastse.g. Saccharomyces, Kluyveromyces and Hansenula) or the α-amylase gene(Bacillus).

By way of example, the secretion of heterologous proteins in E. coli isreviewed in Methods Enzymol (1990) 182:132-43.

Detection

A variety of protocols for detecting and measuring the expression of theamino acid sequence are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays.

A number of companies such as Pharmacia Biotech (Piscataway, N.J.),Promega (Madison, Wis.), and US Biochemical Corp (Cleveland, Ohio)supply commercial kits and protocols for these procedures.

Suitable reporter molecules or labels include those radionuclides,enzymes, fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. No. 3,817,837;U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No.3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 and U.S.Pat. No. 4,366,241.

Also, recombinant immunoglobulins may be produced as shown in U.S. Pat.No. 4,816,567.

Other suitable assays for detecting phytase activity are known in theart and exemplified herein.

Fusion Proteins

The amino acid sequence for use according to the present invention maybe produced as a fusion protein, for example to aid in extraction andpurification. Examples of fusion protein partners includeglutathione-S-transferase (GST), 6×His, GAL4 (DNA binding and/ortranscriptional activation domains) and (β-galactosidase). It may alsobe convenient to include a proteolytic cleavage site between the fusionprotein partner and the protein sequence of interest to allow removal offusion protein sequences.

Preferably, the fusion protein will not hinder the activity of theprotein sequence.

Gene fusion expression systems in E. coli have been reviewed in CurrOpin Biotechnol (1995) 6(5):501-6.

In another embodiment of the invention, the amino acid sequence may beligated to a heterologous sequence to encode a fusion protein. Forexample, for screening of peptide libraries for agents capable ofaffecting the substance activity, it may be useful to encode a chimericsubstance expressing a heterologous epitope that is recognised by acommercially available antibody.

Additional Sequences

The sequences for use according to the present invention may also beused in conjunction with one or more additional proteins of interest(POIs) or nucleotide sequences of interest (NOIs).

Non-limiting examples of POIs include: Xylanase, lipases, acidphosphatases and/or others. These include enzymes that, for example,modulate the viscosity of the feed. The NOI may even be an antisensesequence for any of those sequences.

The POI may even be a fusion protein, for example to aid in extractionand purification or to enhance in vivo phytate metabolism.

The POI may even be fused to a secretion sequence.

Other sequences can also facilitate secretion or increase the yield ofsecreted POI. Such sequences could code for chaperone proteins as forexample the product of Aspergillus niger cyp B gene described in UKpatent application 9821198.0.

The NOI coding for POI may be engineered in order to alter theiractivity for a number of reasons, including but not limited to,alterations, which modify the processing and/or expression of theexpression product thereof. By way of further example, the NOI may alsobe modified to optimise expression in a particular host cell. Othersequence changes may be desired in order to introduce restriction enzymerecognition sites.

The NOI coding for the POI may include within it synthetic or modifiednucleotides—such as methylphosphonate and phosphorothioate backbones.

The NOI coding for the POI may be modified to increase intracellularstability and half-life. Possible modifications include, but are notlimited to, the addition of flanking sequences of the 5′ and/or 3′ endsof the molecule or the use of phosphorothioate or 2′ O-methyl ratherthan phosphodiesterase linkages within the backbone of the molecule.

Antibodies

One aspect of the present invention relates to amino acids that areimmunologically reactive with the amino acid of SEQ ID No. 3.

Antibodies may be produced by standard techniques, such as byimmunisation with the substance of the invention or by using a phagedisplay library.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes but is not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, fragments produced bya Fab expression library, as well as mimetics thereof. Such fragmentsinclude fragments of whole antibodies which retain their bindingactivity for a target substance, Fv, F(ab′) and F(ab′)₂ fragments, aswell as single chain antibodies (scFv), fusion proteins and othersynthetic proteins which comprise the antigen-binding site of theantibody. Furthermore, the antibodies and fragments thereof may behumanised antibodies. Neutralising antibodies, i.e., those which inhibitbiological activity of the substance polypeptides, are especiallypreferred for diagnostics and therapeutics.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with the sequence of the presentinvention (or a sequence comprising an immunological epitope thereof).Depending on the host species, various adjuvants may be used to increaseimmunological response.

Serum from the immunised animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies to thesequence of the present invention (or a sequence comprising animmunological epitope thereof) contains antibodies to other antigens,the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art. In order that such antibodies may bemade, the invention also provides polypeptides of the invention orfragments thereof haptenised to another polypeptide for use asimmunogens in animals or humans

Monoclonal antibodies directed against the sequence of the presentinvention (or a sequence comprising an immunological epitope thereof)can also be readily produced by one skilled in the art and include, butare not limited to, the hybridoma technique Koehler and Milstein (1975Nature 256:495-497), the human B-cell hybridoma technique (Kosbor etal., (1983) Immunol Today 4:72; Cote et al., (1983) Proc Natl Acad Sci80:2026-2030) and the EBV-hybridoma technique (Cole et al., (1985)Monoclonal Antibodies and Cancer Therapy, Alan Rickman Liss Inc, pp77-96).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity may be used (Morrison et al., (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al., (1984) Nature 312:604-608; Takeda etal., (1985) Nature 314:452-454).

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,779) can be adapted to produce thesubstance specific single chain antibodies.

Antibody fragments which contain specific binding sites for thesubstance may also be generated. For example, such fragments include,but are not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W D et al., (1989) Science 256:1275-128 1).

Large Scale Application

In one preferred embodiment of the present invention, the amino acidsequence encoding a C. freundii-derived phytase or the methods of thepresent invention are used for large scale applications. In particular,the methods of the present invention may be used for the large scaleproduction of phytases for industrial use as additives/supplements tofood or feed compositions.

Preferably the amino acid sequence is produced in a quantity of from 5 gper litre to about 10 g per litre of the total cell culture volume aftercultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 100mg per litre to about 900 mg per litre of the total cell culture volumeafter cultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 250mg per litre to about 500 mg per litre of the total cell culture volumeafter cultivation of the host organism.

Use of Phytases

As stated above, the present invention also relates to the production ofphytases as described herein.

In particular, the present invention also relates to the use of theamino acid sequences as disclosed herein in the production of organicand inorganic phosphate compounds.

Thus, the present invention further relates to the use of the nucleotidesequences encoding phytases in generating expression vectors or systemsfor the expression of the phytases.

In addition, the present invention relates to the use of such expressionvectors or systems in the generation of host cells which expressphytases.

The invention further relates to the use of modified host cells in thegeneration of precursors of organic and inorganic phosphate compounds orin the generation of specific organic phosphate compounds.

Suitable organic and inorganic phosphate compounds include myo-inositolpentakis-, tetrakis-, tris-, bis- and monophosphates.

Suitably, the invention therefore provides a method of producing anorganic phosphate compound comprising treating a phytate with a phytasederived from Citrobacter freundii. Preferably, the method ischaracterised in that the enzyme comprises the amino acid sequencesshown as SEQ ID NOs: 3 or a sequence having at least 75% identity(homology) thereto or an effective fragment, or modified form thereof.Suitably, the organic phosphate is phytate or all possible stereoisomersof myo-inositol di-, tri-, tetra, and pentaphosphates. Other suitableorganic phosphates include inositol-tetraphosphates andinositol-oligophosphates. In a preferred embodiment, the method is an invivo biotechnological process.

Such methods for producing an organic phosphate compound may suitablycomprise the steps of:

-   a) providing a host cell that comprises expressible transgenes    comprising C. freundii phytase;-   b) culturing the transgenic organism under conditions suitable for    expression of the transgene; and-   c) recovering the organic phosphate compound from the culture.

The compounds can be used for a number of applications including inassays for the characterisation of phytases. Some inositol phosphatesare involved as signal molecules in intracellular regulation and can beused research chemicals.

In another aspect there is provided a method for production of food oranimal feed. Animal feed is typically produced in feed mills in whichraw materials are first ground to a suitable particle size and thenmixed with appropriate additives. The feed may then be produced as amash or pellets; the later typically involves a method by which thetemperature is raised to a target level and then the feed is passedthrough a die to produce pellets of a particular size. Subsequentlyliquid additives such as fat and enzyme may be added. The pellets areallowed to cool prior to transportation. Production of animal feed mayalso involve an additional step that includes extrusion or expansionprior to pelleting.

Accordingly, the invention further provides the use of an amino acidsequence encoding a phytase or a host cell expressing a phytase toproduce a phytase for use in the manufacture of a food or feed product.In one aspect, there is provided a use of an amino acid sequence asdescribed herein in the manufacture of a food or feed product. Inanother aspect, there is provided a use of a host cell in accordancewith the invention in the manufacture of a food or feed product. Inanother aspect, there is provided a use of an expression vector orsystem in accordance with the invention in the manufacture of a food orfeed product.

The present invention also covers using the enzymes as a component offeed combinations with other components to deliver to animals.

Combination with Other Components

The enzymes of the present invention may be used in combination withother components or carriers.

Suitable carriers for feed enzymes include wheat (coarsely ground). Inaddition there are a number of encapsulation techniques including thosebased on fat/wax coverage, adding plant gums etc.

Examples of other components include one or more of: thickeners, gellingagents, emulsifiers, binders, crystal modifiers, sweetners (includingartificial sweeteners), rheology modifiers, stabilisers, anti-oxidants,dyes, enzymes, carriers, vehicles, excipients, diluents, lubricatingagents, flavouring agents, colouring matter, suspending agents,disintegrants, granulation binders etc. These other components may benatural. These other components may be prepared by use of chemicaland/or enzymatic techniques.

As used herein the term “thickener or gelling agent” as used hereinrefers to a product that prevents separation by slowing or preventingthe movement of particles, either droplets of immiscible liquids, air orinsoluble solids.

The term “stabiliser” as used here is defined as an ingredient orcombination of ingredients that keeps a product (e.g. a food product)from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. afood product ingredient) that prevents the separation of emulsions.

As used herein the term “binder” refers to an ingredient (e.g. a foodingredient) that binds the product together through a physical orchemical reaction.

The term “crystal modifier” as used herein refers to an ingredient (e.g.a food ingredient) that affects the crystallisation of either fat orwater.

“Carriers” or “vehicles” mean materials suitable for compoundadministration and include any such material known in the art such as,for example, any liquid, gel, solvent, liquid diluent, solubiliser, orthe like, which is non-toxic and which does not interact with anycomponents of the composition in a deleterious manner

Examples of nutritionally acceptable carriers include, for example,grain, water, salt solutions, alcohol, silicone, waxes, petroleum jelly,vegetable oils, and the like.

Examples of excipients include one or more of: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar andhigh molecular weight polyethylene glycols.

Examples of disintegrants include one or more of: starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium and certain complex silicates.

Examples of granulation binders include one or more of:polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

Examples of lubricating agents include one or more of: magnesiumstearate, stearic acid, glyceryl behenate and talc.

Examples of diluents include one or more of: water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g they may be delivered by different routes).

As used herein the term “component suitable for animal or humanconsumption” means a compound which is or can be added to thecomposition of the present invention as a supplement which may be ofnutritional benefit, a fibre substitute or have a generally beneficialeffect to the consumer.

By way of example, the components may be prebiotics such as alginate,xanthan, pectin, locust bean gum (LBG), inulin, guar gum,galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),lactosucrose, soybean oligosaccharides, palatinose,isomalto-oligosaccharides, gluco-oligosaccharides andxylo-oligosaccharides.

Food or Feed Substance

The compounds may be used as—or in the preparation of—a food or feedsubstance. Here, the term “food” is used in a broad sense—and coversfood and food products for humans as well as food for animals (i.e. afeed). The term “feed” is used with reference to products that are fedto animals in the rearing of livestock. In a preferred aspect, the foodor feed is for consumption by monogastric animals such as pig, poultryand fish.

The food or feed may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

Food and Feed Ingredients and Supplements

The compounds may be used as a food or feed ingredient.

As used herein the term “food or feed ingredient” includes aformulation, which is or can be added to foods or foodstuffs andincludes formulations which can be used at low levels in a wide varietyof products.

The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The compounds may be—or may be added to—food supplements.

Foods and Feed Compositions

Feed compositions for monogastric animals typically include compositionscomprising plant products which contain phytate. Such compositionsinclude cornmeal, soybean meal, rapeseed meal, cottonseed meal, maize,wheat, barley and sorghum-based feeds.

The phytases described herein may be—or may be added to—foods or feedcompositions.

The present invention also provides a method of preparing a food or afeed ingredient or supplement, the method comprising admixing phytasesproduced by the process of the present invention or the compositionaccording to the present invention with another food ingredient. Themethod for preparing or a food ingredient is also another aspect of thepresent invention. Methods for preparing animal feed are set out above.The enzyme can be added also in the form of a solid formulation, or as afeed additive, such as a pre-mix. A solid form is typically added beforeor during the mixing step; and a liquid form is typically added afterthe pelleting step.

Pharmaceutical

The phytases of the present invention may also be used in pharmaceuticalpreparations or for combination into food stuffs in order to providesome pharmaceutical effect. For example, EP 1,389,915 describes the useof a phytase in a food or drink for increasing the availability ofCalcium, Iron and/or Zinc of the food or drink for humans

In addition, EP 1,392,353 describes a medicament or nutritionalsupplement containing phytase, which is useful for increasingbioavailability of bioelements, e.g., calcium and iron, and forcombating deficiency diseases.

Here, the term “pharmaceutical” is used in a broad sense—and coverspharmaceuticals and/or nutraceuticals for humans as well aspharmaceuticals and/or nutraceuticals for animals (i.e. veterinaryapplications). In a preferred aspect, the pharmaceutical is for humanuse and/or for animal husbandry.

The pharmaceutical can be for therapeutic purposes—which may be curativeor palliative or preventative in nature. The pharmaceutical may even befor diagnostic purposes.

When used as—or in the preparation of—a pharmaceutical, the productand/or the compounds of the present invention may be used in conjunctionwith one or more of: a pharmaceutically acceptable carrier, apharmaceutically acceptable diluent, a pharmaceutically acceptableexcipient, a pharmaceutically acceptable adjuvant, a pharmaceuticallyactive ingredient.

The pharmaceutical may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

Pharmaceutical Ingredient

The product and/or the compounds of the present invention may be used aspharmaceutical ingredients. Here, the product and/or the composition ofthe present invention may be the sole active component or it may be atleast one of a number (i.e. 2 or more) active components.

The pharmaceutical ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The pharmaceutical ingredient may be in the form of an effervescentproduct to improve the dissolving properties of the pharmaceutical.

Forms

The product and/or the compounds of the present invention may be used inany suitable form—whether when alone or when present in a composition.Likewise, phytases produced in accordance with the present invention(i.e. ingredients—such as food ingredients, functional food ingredientsor pharmaceutical ingredients) may be used in any suitable form.

Suitable examples of forms include one or more of: tablets, pills,capsules, ovules, solutions or suspensions, which may contain flavouringor colouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications.

By way of example, if the product and/or the composition are used in atablet form—such as for use as a functional ingredient—the tablets mayalso contain one or more of: excipients, disintegrants, granulationbinders, or lubricating agents.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, carotenoid cleavage compoundsmay be combined with various sweetening or flavouring agents, colouringmatter or dyes, with emulsifying and/or suspending agents and withdiluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

The forms may also include gelatin capsules; fibre capsules, fibretablets etc.

General Recombinant DNA Methodology Techniques

The present invention employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, microbiology, recombinantDNA and immunology, which are within the capabilities of a person ofordinary skill in the art. Such techniques are explained in theliterature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.(1995 and periodic supplements; Current Protocols in Molecular Biology,ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

EXAMPLES

The invention is now further illustrated in the following non-limitingexamples.

Example 1 Phytase Activity Assay

Phytase assays were carried out in microtitre plates. The reactionmixture (100 μl) contained: 2 mM phytate and 0.8 mM CaCl₂ in 200 mMsodium acetate buffer, pH 3.5. The reaction was allowed to proceed for 1h at 37° C. after which time the released phosphate was measured by amodification of a known procedure (Heinonen J. K., Lahti R. J. AnalBiochem. 113 (2), 313-317 (1981)). Briefly, 200 μl of a freshly preparedAMM solution (7.5 N H₂SO₄, 15 mM ammonium molybdate and acetone—1:1:2)was added to the 100 μl reaction mixture in each microtitre plate well.The absorbance at 390 nm was measured not earlier than 10 min and notlater than 30 min after addition of the AMM reagent. The amount ofphosphate was determined by building a calibration curve with phosphatesolutions of known concentrations. For assaying phytase activity atdifferent pH values the following (all 200 mM) buffers were used:glycine/HCl between pH 2.0 and 3.0, sodium acetate/acetic acid betweenpH 3.5 and 5.5, Tris/maleic acid between pH 6.0 and 7.5.

Example 2 Phytase-Producing Strain P3-42

Bacterial strain P3-42 was originally isolated from a mass of decayingbirch leaves collected in a wet forest in southern Finland. The straincan be aerobically cultivated at 30° C. on many simple culture mediae.g. LB (1% peptome, 0.5% yeast extract, 1% NaCl, pH 7.4) or lowphosphate medium PP1 (1% peptone, 1%, beef extract, 0.5%, yeast extract,CaCl₂-0.2M. The medium is adjusted to pH 11 with NaOH and boiled for 10min. The precipitate is removed by filtration, pH re-adjusted to 5.5 andthe medium sterilised by autoclaving for 15 min at 121° C.).

After growth in liquid PP1 medium the strain was found to exhibitphytase activity both at pH 3.5 and 5.5 (assayed as described in Example1). The ratio of activities at 3.5 and 5.5 was about 1.5. The activitywas also measured separately in the cells and culture supernatant ofP3-42. According to these measurements about 90% of all phytase activitywas found in supernatant. The strain was deposited with NCIMB on 22 Sep.2004 under accession number NCIMB 41247.

Example 3 Isolation of Chromosomal DNA from the Strain P3-42

Chromosomal DNA was prepared essentially by the standard procedure(Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1996). A 250 ml culture grown overnight at 30° C. in LBmedium was centrifuged at 10,000 rpm for 30 min, washed in 20 ml of 50mM tris-HCl, 5 mM EDTA pH 8 and re-suspended in 10 ml of cold TES (50 mMtris-HCl, 5 mM EDTA, 15% glucose pH 8). Lysozyme was added to 10 mg/ml,and the cell suspension was incubated at 37° C. for 30-60 min untillysis occurred, ascertained for by dilution of 100 μl of the reactionmixture into 1 ml of 1% SDS and checking for a “slimy” consistency. Atthis time, SDS and Proteinase K (Sigma) were added to a finalconcentration of 1% and 0.5 mg/ml respectively. The reaction mixture wasincubated for 30 min at 56° C. followed by addition of 2 ml of 5 M NaCland 1.4 ml 10% cetyltrimethylammonium bromide (Sigma). The incubationwas continued for 15 min 65° C. The solution was extracted once withchloroform/isoamyl alcohol (24:1) and once with phenol/chloroform. Afterthe extractions, the water phase was mixed with 0.6 vol of isopropanol,the DNA precipitate collected by centrifugation (10,000 rpm, 15 min),washed with 70% ethanol, vacuum dried and re-suspended in 2 ml of 10 mMtris-HCl, 1 mM EDTA pH 8, 5 μg/ml. RNAse.

Example 4 Taxonomic Identification of the Bacterial Strain P3-42

A fragment of the 16S rRNA gene of the strain P3-42 was amplified by thepolymerase chain reaction (PCR) with Taq DNA polymerase (Roche) usingthe primers; 536f (CAGCMGCCGCGGTAATWC) (SEQ ID No. 4) and 1392r(ACGGGCGGTGTGTRC) (SEQ ID No. 5), (Lane, D. J. In Nucleic acidtechniques in bacterial systematics, Stackbrandt, E. and Goodfellow, M.eds, John Wiley & Sons, New York: pp 115-117 (1991)). The followingprogram was used: 1) initial DNA denaturation step of 5 min at 95° C.;2) 30 cycles of 1 min at 94° C., 1 min at 55° C., 1 min at 72° C.; 3) afinal extension step of 70° C. for 10 min. The PCR products,approximately 900 base pairs in size, were purified by electrophoresisin a 0.8% agarose gel and extracted from the gel using a PCRPurification Kit (Qiagen) according to the manufacturer's instructions.The purified PCR products were sequenced by Medprobe (Norway) as acommercial service. The sequenced area is listed as SEQ ID No 1. Thissequence was compared to DNA sequences in the GenBank database(http://www.ncbi.nlm.nih.gov/blast/). The highest match (823 out of 824nucleotides, 99.9%) was found with the sequence of 16S RNA gene fromCitrobacter freundii DSM 30039. Therefore, strain P3-42 can betaxonomically classified as Citrobacter freundii.

Example 5 Cloning of the Phytase Gene from C. freundii P3-42

Chromosomal DNA from the Citrobacter freundii strain P3-42 was partiallydigested with restriction endonuclease Sau3A and the digest fractionatedon 1% agarose gel. The DNA fragments of 3 to 5 kb were isolated from thegel using a gel purification kit (Qiagen) and ligated with BamHIdigested dephosphorylated λ-ZAP arms (Stratagene). Subsequent steps forlibrary construction followed the instructions of Stratagene's ZAPExpress Predigested Vector/Gigapack Cloning Kit. The phage form of thelibrary was converted into a plasmid form by the “mass excision”procedure as described by the manufacturer (Stratagene). Screening ofplasmid library was done by similarly to the earlier published methodsfor the detection of phytase activity on Petri plates (Howson and Davis.Enzyme Microb. Technol. 5, 377-382 (1983); Chen J. C. Biotechnologytechniques 12 (10) 751-761 (1998); Riccio M. L. et al, J. Appl.Microbiol. 82, 177-185 (1997)). Several phytase-positive clones wereisolated and purified by sub-cloning. These isolates were grown inliquid culture (LB medium at 30° C. and 200 rpm for about 24 h) andphytase activity was measured (Example 1) in the resulting cellsuspensions. One clone that had the highest phytase activity (about 5U/ml at pH 3.5) was selected for subsequent characterisation. PlasmidDNA was isolated this clone, named pBK(P3-42) and characterised bypartial DNA sequencing of the insert DNA (sequencing service wasobtained from Medprobe (Norway). This sequence comprising the phytasegene is listed as SEQ ID No: 2. The deduced amino acid sequence of theC. freundii phytase is listed as SEQ ID No: 3. Comparison of the SEQ IDNo: 3 with the sequences in GenBank using the BLAST service provided byNCBI identifies the phytase from E. coli as the closest known homologueof the C. freundii phytase. However, the level homology is low—onlyabout 62% of amino acid residues are identical in both proteins.

Example 6 Amplification and Expression of Phytase Gene from C. freundiiP3-42

Phytase gene was amplified by PCR. Chromosomal DNA of the strain C.freundii P3-42 was used as template and oligonucleotides o42-5(GGAATTCATATGAGTACATTCATCATTCG) (SEQ ID No. 6) and o42-3(GGAATTCGGATCCCTTATTCCGTAACTGCACAC) (SEQ ID No. 7) as primers. Theamplification was carried out using the Expand High Fidelity PCR Systemkit (Roche). The following program was used: 1) initial DNA denaturationfor 3 min at 94° C.; 2) 35 cycles of 45 sec at 94° C., 45 sec at 55° C.,1 min at 68° C., 1 min at 72° C., 1 min at 74° C.; 3) a final extensionstep of 10 min at 72° C. The resulting PCR product was purified byelectrophoresis in a 0.8% agarose gel followed by DNA extraction fromthe gel using a Gel Purification Kit (Qiagen). The purified PCR productwas digested with the restriction enzymes NdeI and BamHI and isolatedfrom the reaction mixture by the PCR Purification Kit (Qiagen). Thevector plasmid pET11a (Novagen) was digested with the restrictionendonucleases NdeI and BamHI, de-phosphorylated using shrimp alkalinephosphatase (Roche) and purified by electrophoresis in a 0.8% agarosegel. The linearised plasmid DNA band was excised from the gel andpurified using a Gel Purification Kit (Qiagen). The two purified DNAfragments were ligated using T4 DNA ligase (Roche). The ligationreaction was precipitated with 70% ethanol, washed with ethanol andre-suspended directly into 50 μl of electrocompetent E. coli XL1-BlueMRF′ cells. The suspension was transferred to a 0.1 cm electroporationcuvette (BioRad) and electroporated using a Gene Pulser Xcell (BioRad)set at 1800 V, 25 μF and 200SΩ. Immediately after electroporation 1 mlof LB medium was added, the cell suspension was transferred to a 15 mlplastic tube (Falcon) and incubated at 37° C. with shaking (200 rpm) for1 hr. The transformed cells were plated onto LB plates containing 100μg/ml ampicillin and incubated overnight at 37° C. 24 transformants weregrown in liquid culture and the cultures used for assaying phytaseactivity and isolation of plasmid DNA. One clone producing highestphytase activity and generating the expected restriction pattern of theplasmid DNA was selected. The plasmid contained by this clone namedpET11(P3-42) was used to transform the expression host strainBL21(DE3)pLysS (Novagen). The transformed cell suspension, was shakenfor 1 h at 37° C. in LB containing 2% glucose and inoculated into 50 mlof LB containing ampicillin (100 μg/ml) and glucose (2%) and grownovernight at 30° C. with shaking (200 rpm). The OD of the resultingculture was measured at 600 nm and the culture was used to inoculate 1 lof LB+ampicillin (100 μg/ml) to an OD₆₀₀ of 0.04. Growth was continuedovernight at 30° C. The phytase activity in such cultures was typically50-60 U/ml (measured at pH 3.5). Almost all of the phytase was secretedinto the culture medium. The fact that C. freundii phytase is anefficiently secreted enzyme both in its native host and in duringheterologous expression in E. coli is in contrast to the intracellularnature of a phytase from C. brakii (Kim H. W. et al. Biotechnol. Lett.25, 1231-1234 (2003)). The activity in the culture of a control strainBL21(DE3)pLysS transformed with pET11 grown under the same conditionswas below 0.05 U/ml.

Example 7 Purification of the Recombinant Phytase from C. freundii P3-42

The culture of BL21(DE3)pLysS transformed with pET11(P3-42) wascentrifuged to remove the bacterial cells, concentrated using a rotaryevaporator to about 1/10 of the original volume and dialysed againstwater until the conductivity of the solution decreased below 250 μS/cm.The pH of the solution was adjusted to 8.0 with tris base and it wasapplied to a column (3×20 cm) of DEAE Sepharose Fast Flow (AmershamBiosciences) equilibrated with 25 mM tris-HCl, pH 8.0. The column waswashed with the equilibration buffer at a flow rate of 3 ml/min for 30min followed by elution with three successive gradients of NaCl in 25 mMtris-HCl, pH 8.0: 0-50 mM, 50-150 mM and 150-500 mM. Each of the threegradients was programmed for 1 h with a constant flow rate of 3 ml/min 9ml fractions were collected and assayed for phytase activity. One strongpeak of activity was detected. The protein in the peak fraction wasconcentrated using Centriplus concentrators (Amicon) and analysed SDSPAGE using a 12% gel and the standard Laemmli buffer system. The resultsof this analysis indicated that the preparation of recombinant C.freundii P3-42 phytase obtained by DEAE Sepharose contains a singleprominent protein component. Semi-quantitative analyses based onscanning of the digital image of the gel (FIG. 1) indicate the purity ofabout 60-70%.

Example 8 pH Profile of the Recombinant Phytase from C. freundii P3-42

Dependence of the activity of the C. freundii P3-42 phytase from(purified according to the Example 7) on pH was studied in buffers andunder conditions described in Example 1. The enzyme was active in abroad pH area (2-5.5) with two activity maxima around pH 3 and 4-4.5(FIG. 2).

Example 9 Substrate Specificity of the Recombinant Phytase from C.freundii P3-42

The fractions of inositol phosphates containing three, four or fivephosphates per inositol residue were isolated by ion-exchangechromatography from a partial hydrolysate of phytic acid treated withfungal phytase (Natuphos). Production and purification of thesepreparations was a commercial service of BioChemis Ltd (St. Petersburg,Russia). Contamination of each fraction with inositol-phosphates havinga different degree of phosphorylation was less that 5% as judged by HPLC(Sandberg A. S., Ahderinne R. J. Food Sci. 51 (3), 547-550). Commercialfructose 1,6-diphosphate and fructose 6-phosphate (Sigma) were used asmodel substrates used to estimate the specificity of the C. freundiiP3-42 phytase towards di- and monophosphate substrates. The activity ofthe C. freundii phytase purified according to the Example 7 withdifferent substrates was measured by the standard assay (Example 1) atpH 3.5 using 2 mM concentrations of substrates in the final reactionmixture. The results (FIG. 3) indicate that that the enzyme has maximumactivity with inositol pentaphosphate. Activities with inositol tri- andtetraphosphates as well as phytic acid were rather similar whilefructose 1,6-diphosphate was a rather poor substrate. Hydrolysis offructose 6-phosphate was below reliable detection limit.

Example 10 Specific Activity of the Recombinant Phytase from C. freundiiP3-42

Specific activity of the C. freundii phytase was estimated using thepurified preparation according to the Example 7. The phytase activitywas measured at pH 3.5 according to the Example 1. Phytase concentrationwas calculated by measuring total protein concentration with BCA ProteinAssay Kit (Pierce) and correcting it by phytase content estimated by SDSPAGE (Example 7). According to these measurements, the specific activityof the recombinant phytase from C. freundii P3-42 is about 1100 U/mg.

Example 11 Comparison of C. freundii P3-42 Phytase with the Phytase fromC. brakii YH-15

The only phytase from a bacterium belonging to the Citrobacter familydescribed earlier is the intracellular phytase from C. brakii YH-15 (KimH. W. et al. Biotechnol. Lett. 25, 1231-1234 (2003)). This enzyme sharessome properties with the secreted phytase of C. freundii of the presentinvention both enzymes are acid phytases of high specific activity.Direct comparison of the amino acid sequences of the two enzymes isimpossible because the sequence information regarding the C. brakiienzyme is limited to a stretch of 10 amino acid residues. The deducedamino acid sequence of C. freundii phytase contains a fragment sharing 9out of 10 residues with the sequence from C. brakii enzymes. However,comparison of such short fragments of sequence does not allow anyconclusions about the overall homology of the two enzymes to be made.The most striking difference between the two enzymes is in cellularlocation: while the enzyme from C. brakii is intracellular, the C.freundii phytase is clearly a secreted enzyme. The enzyme is secreted inits native host, its deduced amino acid sequence does contain a signalpeptide (as predicted by the Signal P algorithm:http://www.cbs.dtu.dk/services/SignalP/), the enzyme is also veryefficiently secreted from E. coli under its native signal peptide. Inaddition to that, there are a number of significant differences inbiochemical properties of the two enzymes (Table 1). Table 1 Comparisonof the phytase from C. freundii P3-42 with phytase from C. brakii YH-14.

Property C. brakii YH-15 phytase C. freundii P3-42 phytase LocalisationIntracellular Secreted Specific activity 3457 U/mg (pH 4) 1100 U/mg (pH3.5) pH optimum 4.0 3.0, 5.0 Thermostability 20%⁽*⁾ 58% ⁽*⁾Measuredunder conditions described by Kim et al. (Biotechnol. Lett. 25,1231-1234 (2003)): heat treatment in 100 mM Na acetate, pH 4, 60° C., 30min followed by standard assay at 37° C.

Example 12 Generation and Characterisation of Phytase Variants

Phytase variants were constructed by mutagenesis of the sequence SEQ IDNo. 2 using mutagenesis methods as listed above such as the methodsdisclosed in Morinaga et al (Biotechnology (1984)2, p 646-649), or inNelson and Long (Analytical Biochemistry (1989), 180, p 147-151), or theError Threshold Mutagenesis protocol described in WO 92/18645.

Phytase enzyme variants were characterized after heterologous expressionin one or more of the following expression hosts: Escherichia coli K12;Bacillus subtilis; Saccharomyces cerevisiae.

1. Thermostability

The thermostability of the variants was characterized by theinactivation temperature of the enzyme. The inactivation temperature wasdetermined by measuring the residual activity of the enzyme in an enzymeassay as described in Example 1 after incubation for 10 min at differenttemperatures and subsequent cooling to room temperature. Theinactivation temperature is the temperature at which the residualactivity is 50% compared to the residual activity after incubation forthe same duration under the same conditions at room temperature. Whereappropriate interpolations and extrapolations from the measured activitydata are done in order to determine the temperature corresponding to 50%residual activity. Thermostability differences in ° C. were calculatedby subtracting the inactivation temperatures of two enzymes from eachother. (i.e. Thermostability difference (T.D.) is compared to parentphytase (=inactivation temperature (variant)−inactivation temperature(parent))

Table 2 lists the thermostability differences for different variants:

TABLE 2 Thermostability differences for variants derived from the parentphytase P3-42 having the sequence shown in Seq ID No 3. Variant T.D.P229S 1.5 D112V 1.5 Q82R 1.5 Q274H 1.0 D112Y 2.5 F88Y 1.5 K46E 2.0 S233C2.0 R288M 4.0 I384L 1.0 Q385R 1.5 Q274L 2.0 E307Y 1.0 T199I 2.0 Q82K 2.0T203I 1.0 K46E/Q82H 2.5 Q82K/V105I 1.0 N148D/T362I 1.5 K46E/L414I 1.0F88Y/Y136N 1.0 N95P/N96S 1.5 N95P/N96P 2.0 Q97T/T98G 1.0 Y177F/T199I 2.5Q274L/Q370H 3.0 K46E/N96Y 2.5 N148D/L301S 1.5 E24D/R288M 1.5 E140V/A322V2.0 K46E/S195T 2.0 E75K/N365D 1.5 T98P/S235A 2.0 L160F/L215F 1.0Q274L/K395T 1.5 G67R/Q279E/N308T 2.0 K161N/P229S/R288M 2.0D53N/D57Y/M152V 2.0 F122Y/S156T/P229S 1.5 E23K/K46E/Q82H 6.0K46E/Q82H/Q385R 5.0 T203W/E204N/K205R 2.0 T203W/E204H/K205R 3.0T203W/E204R/K205R 3.0 T203W/E204A/K205R 3.0 A22T/K151G/N308D 2.0E23K/E75K/F88Y 2.0 M152K/N225D/L301S 2.0 S78T/Q274L/S408I 2.0L176Q/T199I/T366S 1.5 K46E/V77I/T203S 3.0 K46R/T199I/D367N 1.5G74R/E204G/R288M 1.5 A22T/T199I/S206T/T207A 1.5 Q82R/F88Y/L126I/I384L3.0 K46E/Q82H/E168D/Q274L 5.0 Q82K/T154I/Q279E/N308T 5.5Q82R/D112V/Q274H/T362A 5.0 E24D/E79V/N95D/K360N 1.0E23K/M28L/A109T/T143P/I384L 2.0 D53N/D57Y/T199I/P229S/R288M 6.0K46E/Q82H/N148D/T154I/T362I 7.0 D53N/D57Y/P229S/R288M/K358R 5.5D53N/D57Y/T154I/P229S/R288M 7.0 Y136N/T199I/T203L/E204I/K205P 3.0E23Q/S101F/Q274L/I384M/K391N 2.0 K46E/Q82H/N95D/D112V/K142R/D383V 5.5D53N/D57Y/M152V/P229S/R288M/A393P 7.0 D53K/D57Y/M152V/P229S/R288M/A393P8.0 D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T 6.5D53N/D57Y/M152V/E204V/P229S/R288M/A393P 8.0D53N/D57Y/M152V/T154I/P229S/R288M/A393P 8.0D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P 8.5K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P 8.0Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P 9.0Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P 9.0D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P 7.5Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/ 10.0 A393P/D397ND53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/ 10.0N164D/Q274H/T362I/I384L/A393PD53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/ 10.0Y177F/Q274H/Q279E/T362I/A393PD53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/ 9.0Q274L/G343A/T362I/I384L/A393P

2. Other Characteristics

Other characteristics were also improved.

Thermostability, specific activity, and pepsin stability of selectedvariants were compared using assays as described above. The pepsinstability of such variants was characterized by residual activitiesmeasured at pH 3.5, 37° C. after pepsin incubation compared to controlconditions (residual activity=activity after pepsin incubation/activityafter incubation under control conditions). The pepsin incubation wasperformed for 2 hours at pH 2.0, 0.25 mg/ml pepsin, 1 mM CaCl₂ and 5mg/ml BSA at 37° C. Control conditions were 2 hours at pH 5.0, 1 mMCaCl₂ and 5 mg/ml BSA at 37° C.

Table 3 shows properties of selected variants (derived from and comparedto wt phytase according to Seq ID No. 3).

Specific Pepsin stability activity [% [% residual activity] Variant T.D.[° C.] of wt activity] (wt = 41%) K46E/ 2.2 96 65 Q82H

Sequence Information

SEQ ID No: 1 CGATTACTAGCGATTCCGACTTCTGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGTTCCCGAAGGCACCAAAGCATCTCTGCTAAGTTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTAC SEQ ID No 2:AAAGGTGGTGCTGGTAATGAGTACATTCATCATTCGTTTATTATTTTTTTCTCTCTTATGCGGTTCTTTCTCAATACATGCTGAAGAGCCGAACGGTATGAAACTTGAGCGGGTTGTGATAGTGAGCCGTCATGGAGTAAGAGCACCTACGAAGTTCACTCCAATAATGAAAGATGTTACACCCGATCAATGGCCACAATGGGATGTGCCGTTAGGATGGCTAACGCCTCGTGGGGGAGAACTTGTTTCTGAATTAGGTCAGTATCAACGTTTATGGTTCACAAGCAAAGGTCTGTTGAATAATCAAACGTGCCCATCTCCAGGGCAGGTTGCTGTTATTGCAGACACGGATCAACGCACCCGTAAAACGGGTGAGGCGTTTCTGGCTGGGTTAGCACCAAAATGTCAAATTCAAGTGCATTATCAGAAGGATGAAGAAAAAACTGATCCTCTTTTTAATCCAGTAAAAATGGGGACATGTTCGTTTAACACATTGAAGGTTAAAAACGCTATTCTGGAACGGGCCGGAGGAAATATTGAACTGTATACCCAACGCTATCAATCTTCATTTCGGACCCTGGAAAATGTTTTAAATTTCTCACAATCGGAGACATGTAAGACTACAGAAAAGTCTACGAAATGCACATTACCAGAGGCTTTACCGTCTGAACTTAAGGTAACTCCTGACAATGTATCATTACCTGGTGCCTGGAGTCTTTCTTCCACGCTGACTGAGATATTTCTGTTGCAAGAGGCCCAGGGAATGCCACAGGTAGCCTGGGGGCGTATTACGGGAGAAAAAGAATGGAGAGATTTGTTAAGTCTGCATAACGCTCAGTTTGATCTTTTGCAAAGAACTCCAGAAGTTGCCCGTAGTAGGGCCACACCATTACTCGATATGATAGACACTGCATTATTGACAAATGGTACAACAGAAAACAGGTATGGCATAAAATTACCCGTATCTCTGTTGTTTATTGCTGGTCATGATACCAATCTTGCAAATTTAAGCGGGGCTTTAGATCTTAACTGGTCGCTGCCCGGTCAACCCGATAATACCCCTCCTGGTGGGGAGCTTGTATTCGAAAAGTGGAAAAGAACCAGTGATAATACGGATTGGGTTCAGGTTTCATTTGTTTATCAGACGCTGAGAGATATGAGGGATATACAACCGTTGTCGTTAGAAAAACCTGCCGGCAAAGTTGATTTAAAATTAATTGCATGTGAAGAGAAAAATAGTCAGGGAATGTGTTCGTTAAAAAGTTTTTCCAGGCTCATTAAGGAAATTCGCGTGCCAGAGTGTGCAGTTACGGAATAAGTAACTAATTACTATATATAGCGTATTAAAAAATAGAAACCCCCGGTTTGTAGTCGGGGGTATTCGTATTGTTCATAATTAC A SEQ ID No: 3MSTFIIRLLFFSLLCGSFSIHAEEPNGMKLERVVIVSRHGVRAPTKFTPIMKDVTPDQWPQWDVPLGWLTPRGGELVSELGQYQRLWFTSKGLLNNQTCPSPGQVAVIADTDQRTRKTGEAFLAGLAPKCQIQVHYQKDEEKTDPLFNPVKMGTCSFNTLKVKNAILERAGGNIELYTQRYQSSFRTLENVLNFSQSETCKTTEKSTKCTLPEALPSELKVTPDNVSLPGAWSLSSTLTEIFLLQEAQGMPQVAWGRITGEKEWRDLLSLHNAQFDLLQRTPEVARSRATPLLDMIDTALLTNGTTENRYGIKLPVSLLFIAGHDTNLANLSGALDLNWSLPGQPDNTPPGGELVFEKWKRTSDNTDWVQVSFVYQTLRDMRDIQPLSLEKPAGKVDLKLIACEEKNSQGMCSLKSFSRLIKEIRVPECAVTE

All publications mentioned in the above specification, and referencescited in said publications, are herein incorporated by reference.Various modifications and variations of the described methods and systemof the present invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the present invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1. An isolated polypeptide comprising the amino acid sequencecorresponding to Citrobacter freundii phytase or a homologue, a modifiedform, a functional equivalent or an effective fragment thereof.
 2. Anisolated polypeptide as claimed in claim 1 comprising the amino acidsequence as shown in SEQ ID NO: 3 or a sequence having at least 75%identity (homology) thereto or a functional fragment thereof.
 3. Anisolated polypeptide having the amino acid sequence as set out in SEQ IDNO: 3 or a sequence having at least 75% identity (homology) thereto or afunctional fragment thereof.
 4. A phytase characterised in that it isderived from Citrobacter freundii strain P3-42 deposited under accessionnumber NCIMB
 41247. 5. A phytase or functional equivalent thereofcharacterised in that said phytase has a specific activity of at least1100 U/mg wherein said specific activity is determined by incubatingsaid phytase in a solution containing 2 mM phytate, 0.8 mM CaCl₂ in 200mM sodium acetate buffer at pH 3.5.
 6. A phytase or functionalequivalent thereof characterised in that said phytase has two activitymaxima around pH 3 and pH 4-4.5 wherein said activity is determined byincubating said phytase in a solution containing 2 mM phytate, 0.8 mMCaCl₂ in 200 mM sodium acetate buffer.
 7. An isolated polypeptide orphytase as claimed claim 1 that comprises one or more mutations at thefollowing positions (numbering according to the numbering in SEQ ID No.3): 22, 23, 24, 28, 46, 53, 57, 67, 74, 75, 77, 78, 79, 82, 88, 95, 96,97, 98, 101, 102, 103, 105, 109, 112, 122, 126, 136, 140, 142, 143, 148,151, 152, 154, 156, 160, 161, 164, 168, 170, 176, 177, 195, 199, 203,204, 205, 206, 207, 215, 224, 225, 229, 233, 235, 274, 279, 288, 301,307, 308, 322, 343, 358, 360, 362, 365, 366, 367, 370, 383, 384, 385,386, 391, 393, 395, 397, 408,
 414. 8. An isolated polypeptide or phytaseas claimed in claim 7 wherein said phytase comprises one or more of thefollowing mutations: A22T, E23K, E23Q, E24D, M28L, K46E, K46R, D53K,D53N, D57Y, G67R, G74R, E75K, E75V, V77I, S78T, E79V, Q82H, Q82K, Q82R,F88Y, N95D, N95P, N96P, N96S, N96Y, Q97T, T98G, T98P, S101F, P102L,G103E, V105I, A109T, D112V, D112Y, F122Y, L126I, Y136N, E140V, K142R,T143I, T143P, N148D, K151G, M152K, M152V, T154I, S156T, L160F, K161N,N164D, E168D, A170T, L176Q, L176V, Y177F, S195T, T199I, T203I, T203L,T203S, T203W, E204A, E204G, E204H, E204I, E204N, E204R, E204V, K205P,K205R, S206R, S206T, T207A, T207S, L215F, D224H, N225D, N225E, P229S,S233C, A235A, Q274H, Q274L, Q279E, R288M, L301S, E307Y, N308D, N308T,A322V, G343A, K358R, K360N, T362A, T362I, N365D, T366S, D367N, Q370H,D383V, I384F, I384L, I384M, Q385R, P386Q, K391N, A393P, K395T, D397N,S408I, L414I.
 9. An isolated polypeptide or phytase as claimed in claim7 comprising one mutation selected from the group consisting of: P229S;D112V; Q82R; Q274H; D112Y; F88Y; K46E; S233C; R288M; I384L; Q385R;Q274L; E307Y; T199I; Q82K and T203I.
 10. An isolated polypeptide orphytase as claimed in claim 7 comprising a combination of mutationsselected from the group consisting of: K46E/Q82H; Q82K/V105I;N148D/T362I; K46E/L414I; F88Y/Y136N; T154I/P386Q; N95P/N96S; N95P/N96P;Q97T/T98G; D224H/N225E; Y177F/T199I; Q274L/Q370H; K46E/N96Y;N148D/L301S; E24D/R288M; E140V/A322V; K46E/S195T; E75K/N365D;T98P/S235A; L160F/L215F; Q274L/K395T; G67R/Q279E/N308T;K161N/P229S/R288M; D53N/D57Y/M152V; F122Y/S156T/P229S;T199I/S206R/T207S; E23K/K46E/Q82H; K46E/Q82H/Q385R; T203W/E204N/K205R;T203W/E204H/K205R; T203W/E204R/K205R; T203W/E204A/K205R;A22T/K151G/N308D; E23K/E75K/F88Y; M152K/N225D/L301S; S78T/Q274L/S408I;L176Q/T199I/T366S; K46E/V771/T203 S; K46R/T199I/D367N; G74R/E204G/R288M;A22T/T199I/S206T/T207A; Q82R/F88Y/L126I/I384L; K46E/Q82H/E168D/Q274L;Q82K/T154I/Q279E/N308T; Q82R/D112V/Q274H/T362A; E24D/E79V/N95D/K360N;E23K/M28L/A109T/T143P/I384L; D53N/D57Y/T199I/P229S/R288M;K46E/Q82H/N148D/T154I/T362I; D53N/D57Y/P229S/R288M/K358R;D53N/D57Y/T154I/P229S/R288M; Y136N/T199I/T203L/E204I/K205P;E23Q/S101F/Q274L/I384M/K391N; K46E/Q82H/N95D/D112V/K142R/D383V;D53N/D57Y/M152V/P229S/R288M/A393P; D53K/D57Y/M152V/P229S/R288M/A393P;D53N/D57Y/F88Y/M152V/P229S/Q279E/N308T;D53N/D57Y/M152V/E204V/P229S/R288M/A393P;D53N/D57Y/M152V/T154I/P229S/R288M/A393P;D53N/D57Y/Q82H/G103E/M152V/P229S/R288M/A393P;K46E/D53N/D57Y/T143I/M152V/L176V/P229S/R288M/A393P;Q82K/F88Y/N96P/Q97T/T98G/V105I/Q274H/Q279E/A393P;Q82R/F88Y/N95P/N96P/Q97T/Q279E/I384L/P386Q/A393P;H18Q/D53N/D57Y/E75V/M152V/A170T/P229S/R288M/Q385R/A393P;Q82K/F88Y/N96P/T98G/Y136N/M152V/Y177F/T362I/I384F/A393P/D397N;D53N/D57Y/F88Y/N95P/N96P/V105I/D112V/Y136N/N148D/N164D/Q274H/T362I/I384L/A393P,D53N/D57Y/Q82K/F88Y/N95P/P102L/V105I/Y136N/N148D/Y177F/Q274H/Q279E/T362I/A393P;D53N/D57Y/Q82K/F88Y/N96P/T98G/V105I/D112V/Y177F/Q274L/G343A/T362I/I384L/A393P;11. An isolated nucleic acid molecule coding for the enzyme ofCitrobacter freundii phytase, or a homologue thereof.
 12. An isolatednucleic acid molecule as claimed in claim 11 encoding a polypeptidecomprising the amino acid sequence as shown in SEQ ID NO: 3 or asequence having at least 75% identity (homology) thereto or an effectivefragment thereof.
 13. An isolated nucleic acid molecule comprising anucleotide sequence that is the same as, or is complementary to, orcontains any suitable codon substitutions for any of those of SEQ ID NO:2 or comprises a sequence which has at least 75%, 80%, 85%, 90%, 95% or99% sequence homology with SEQ ID NO:
 2. 14. An isolated nucleic acidmolecule coding for the isolated polypeptide or phytase as claimed inclaim
 1. 15. An isolated nucleic acid molecule comprising the sequenceas set out in SEQ ID NO:
 2. 16. A plasmid or vector system comprising anisolated polypeptide or phytase as claimed in claim 1 or a homologue orderivative thereof.
 17. A plasmid or vector system as claimed in claim16 which comprises a nucleic acid sequence as claimed in claim
 11. 18. Aplasmid or vector system as claimed in claim 16 which comprises anucleic acid sequence as set out in SEQ ID No: 2 or a sequence that isat least 75% homologous thereto or an effective fragment thereof.
 19. Aplasmid or vector system as claimed in claim 16 wherein said plasmid orvector system is an expression vector for the expression of therespective phytase enzyme or homologue, modified form, functionalequivalent or effective fragment thereof, in a host cell or amicroorganism.
 20. A host cell transformed or transfected with a plasmidor vector system as claimed in claim
 16. 21. A host cell as claimed inclaim 20 which comprises a phytase which comprises an amino acidsequence, or functional fragment thereof, as set out in SEQ ID NO: 3, ora sequence that is at least 75% homologous thereto, or a variant thereofaccording to claims
 7. 22. A host cell as claimed in claim 20 whereinsaid host cell is derived from a microorganism including bacteria, suchas B. subtilis, E. coli and fungi, including yeast such as H.polymorpha, S. pombe, S. cerevisiae.
 23. A host cell as claimed in claim22 wherein said microorganism is a prokaryotic bacterial cell and,preferably, E. coli.
 24. A bacterial cell strain Citrobacter freundiiP3-42 deposited under accession number NCIMB
 41247. 25. A method ofproducing a phytase comprising expressing an amino acid sequence as setout in SEQ ID NO: 3, or a sequence having at least 75% homology theretoor a modified form or a variant or an effective fragment thereof in ahost cell and separating the phytase from the host cell culture medium.26. A food or animal feed composition comprising a phytase as claimed inclaim
 1. 27. Use of a phytase as claimed in claim 1 in food or animalfeed.
 28. A method for production of food or animal feed comprising astep of spraying a phytase as claimed in claim 1 in liquid form ontosaid food or animal feed.
 29. A method for production of food or animalfeed comprising a step of mixing the phytase as claimed in claim 1 as adry product with said food or animal feed.
 30. A method of preparing aphytase enzyme variant, which method comprises: a) selecting a parentphystase enzyme, wherein the parent phystase enzyme is selected from: i.a parent phytase enzyme with at least 75% homology to SEQ ID NO 3 ii. aparent phytase enzyme derived from Citrobacter spp. b) making at leastone alteration which is an insertion, a deletion or a substitution of anamino acid residue in the parent phytase enzyme to obtain a phytaseenzyme variant c) screening for a phytase enzyme variant which comparedto the parent phytase enzyme has: i. higher thermal stability and/or ii.specific activity and/or iii. proteolytic stability d) preparing thephytase enzyme variant.
 31. A method of preparing a phytase enzymevariant, which method comprises: a) subjecting DNA sequence encoding aparent phytase enzyme to mutagenesis, wherein the parent phytase isselected from i. a parent phytase enzyme with at least 75% homology toSEQ ID NO 3 ii. a parent phytase enzyme derived from Citrobacter spp. b)expressing the mutated DNA sequence obtained in strep (A) in a hostcell, and c) screening for host cells expressing a phytase enzymevariant which compared to the parent phytase enzyme has: i. higherthermal stability and/or ii. higher specific activity and/or iii. higherproteolytic stability d) preparing the phytase enzyme variant expressedby the host cell.